Engineered natural killer (nk) cells and compositions and methods thereof

ABSTRACT

The present invention provides engineered Natural Killer (NK) cells and methods of producing engineered NK cells. The engineered NK cells and compositions containing the engineered NK cells are useful for treating diseases such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application No.62/457,098 filed Feb. 9, 2017, entitled “ENGINEERED NATURAL KILLER (NK)CELLS AND COMPOSITIONS AND METHODS THEREOF,” and U.S. provisionalapplication No. 62/484,350 filed Apr. 11, 2017, entitled “ENGINEEREDNATURAL KILLER (NK) CELLS AND COMPOSITIONS AND METHODS THEREOF,” thecontents of which are incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled776032000140SeqList.txt, created Feb. 6, 2018, which is 12,126 bytes insize. The information in the electronic format of the Sequence Listingis incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides engineered Natural Killer (NK) cells andmethods of producing engineered NK cells. The engineered NK cells andcompositions containing the engineered NK cells are useful for treatingdiseases such as cancer.

BACKGROUND OF THE INVENTION

NK cells were discovered 40 years ago by their ability to recognize andkill tumor cells without the requirement of prior antigen exposure.Since then, NK cells have been seen as promising agents for cell basedcancer therapy. Most cancers lack identifiable, tumorspecific antigensin the HLA context, and thus cannot succumb to antigen specificcytotoxic T lymphocytes. Since a wide range of cancer cells aresensitive to NK cytotoxicity, one major advantage of using NK cells isthat transplantation of natural killer (NK) cell can be employed againstcancer cells in an allogeneic setting, without risk of graft-versus-hostdisease. The therapeutic promise of NK cells has been limited by thedifficulty in obtaining a theoretically effective amount of NK cells,particularly NK cells that exhibit cytotoxic effector functions forkilling malignant tumor cells or infected cells. Accordingly, there is aneed for engineered NK cells for therapeutic use. Provided herein areembodiments that meet such needs.

BRIEF SUMMARY OF THE INVENTION

Provided herein are engineered natural killer (NK) cells that havereduced expression, activity and/or signaling of the FcRγ chain. In someembodiments, the provided engineered cells exhibit enhanced immuneresponses, including for the treatment of cancer, microbial infectionsand/or viral infections in a subject. Also provided are methods oftreatment of a subject by administering a therapeutically effectiveamount of the provided engineered NK cells. In some embodiments, theengineered NK cells are useful for enhancing therapeutic responses to anadministered therapeutic antibody, such as to an anti-cancer, anti-viralor anti-microbial monoclonal antibody.

In some embodiments, provided herein is an engineered NK cell, whereinthe NK cell is genetically engineered to reduce FcRγ chain expression,activity and/or signaling in the cell. In some of these embodiments, theengineered NK cell comprises a genetic disruption of a gene resulting inreduced FcRγ chain expression, activity and/or signaling in the cell. Insome cases, the genetic disruption can result in a deletion or mutationof the gene. In some embodiments, the engineered NK cell comprises aninhibitory nucleic acid that reduces expression of a gene that resultsin reduced FcRγ chain expression, activity and/or signaling in the cell.

In some embodiments, the engineered NK cell comprises a geneticdisruption of a gene encoding FcRγ chain and/or a genetic disruptionresulting in reduced expression of FcRγ chain in the engineered NK cell.In some embodiments, the engineered NK cell comprises a geneticdisruption of a gene encoding a protein regulating expression oractivity of FcRγ chain and/or a genetic disruption resulting in reducedexpression of a protein regulating expression or activity of FcRγ chain.In some embodiments, the engineered NK cell comprises a geneticdisruption of a gene encoding a protein involved in FcRγ chain-dependentsignaling and/or a genetic disruption resulting in reduced expression ofa protein involved in FcRγ chain-dependent signaling. In someembodiments, the engineered cell can contain one or more of the abovegenetic disruptions. In some embodiments, the genetic disruptioncomprises a deletion, mutation and/or insertion resulting in a prematurestop codon in the gene or a frameshift of the open reading frame of thegene. In some embodiments, both alleles of the gene encoding FcRγ chain,the gene encoding a protein regulating expression or activity of FcRγchain and/or the gene encoding a protein involved in FcRγchain-dependent signaling are disrupted in the engineered NK cells.

In some embodiments, the engineered NK cell comprises an inhibitorynucleic acid molecule targeting a gene in the NK cell resulting inreduced expression of FcRγ chain, reduced expression of a proteinregulating expression or activity of FcRγ chain and/or reducedexpression of a protein involved in FcRγ chain-dependent signaling. Insome embodiments, the inhibitory nucleic acid interferes with or reducesexpression of a gene encoding FcRγ chain, a gene encoding a proteinregulating expression or activity of FcRγ chain and/or a gene encoding aprotein involved in FcRγ chain-dependent signaling. In some embodiments,the inhibitory nucleic acid comprises an RNA interfering agent. In someembodiments, the inhibitory nucleic acid comprises siRNA, shRNA, ormiRNA.

In some of any such embodiments, the expression of FcRγ chain, a proteinregulating expression or activity of FcRγ chain and/or a proteininvolved in FcRγ chain-dependent signaling is reduced by greater than orgreater than about 50%, 60%, 70%, 80%, 90%, or 95% in the engineered NKcell as compared to the expression of the protein in the NK cell that isnot genetically engineered.

In some of any such embodiments, expression of a protein regulatingexpression or activity of FcRγ chain is reduced in the engineered NKcell. In some of these embodiments, the protein regulating expression oractivity of FcRγ chain is a transcription factor. In some embodiments,the transcription factor is PLZF (ZBTB16) or HELIOS (IKZF2).

In some of any such embodiments, the expression of a protein involved inFcRγ chain-dependent signaling is reduced in the engineered NK cell. Insome embodiments, the protein involved in FcRγ chain-dependent signalingis a downstream signaling molecule of FcRγ. In some of theseembodiments, the downstream signaling molecule is SYK, DAB2 or EAT-2.

In some of any such embodiments, expression of FcRγ chain is reduced inthe engineered NK cell. In some embodiments, the engineered NK cellcomprises a genetic disruption in a gene encoding FcRγ chain. In some ofthese embodiments, the genetic disruption comprises a deletion, mutationand/or insertion resulting in a premature stop codon in the gene or aframeshift of the open reading frame of the gene. In some embodiments,both alleles of the gene encoding FcRγ chain are disrupted in the genomeof the engineered NK cell. In some embodiments, the engineered NK cellcomprises an inhibitory nucleic acid molecule that targets a geneencoding FcRγ chain, thereby reducing expression of FcRγ chain in thecell. In some embodiments, the inhibitory nucleic acid moleculecomprises a sequence complementary to the gene encoding FcRγ chain. Insome embodiments, the inhibitory nucleic acid comprises an RNAinterfering agent In some embodiments, the inhibitory nucleic acidcomprises siRNA, shRNA, or miRNA. In some embodiments, the expression ofFcRγ chain is reduced by greater than or greater than about 50%, 60%,70%, 80%, 90%, or 95% in the engineered NK cell as compared to theexpression in the NK cell that is not genetically engineered.

In some of any of the provided embodiments, the reduced FcRγ chainexpression, activity and/or signaling in the engineered NK cell ispermanent, transient or inducible. In some embodiments, the expression,activity and/or signaling of FcRγ chain is reduced by greater than orgreater than about 50%, 60%, 70%, 80%, 90%, or 95% as compared to theexpression, activity and/or signaling in the NK cell that is notgenetically engineered. In some embodiments, the expression of FcRγchain expressed in the cell is undetectable in an immunoblot assay.

In some of any of the embodiments, the engineered NK cell is derivedfrom a primary cell obtained from a subject. In some embodiments, thesubject is human.

In some of any of the embodiments, the engineered NK cell is derivedfrom a clonal cell line. In some embodiments, the clonal cell line isNK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1.

In some of any of the embodiments, CD16 is expressed on the surface ofthe engineered NK cell. In some of any of the embodiments, theengineered NK cell expresses CD3-zeta (CD3ζ) chain. In some embodiments,the expressed CD16 and/or CD3ζ chain is endogenous to the NK cell. Insome embodiments, the engineered NK cell is additionally engineered toexpress a recombinant or heterologous CD16 and/or CD3ζ chain in the NKcell. In some embodiments, the engineered NK cell comprises arecombinant or heterologous CD16 gene and/or a recombinant orheterologous CD3-zeta (CD3ζ) chain. In some embodiments, the recombinantor heterologous CD16 comprises a CD16-activating mutation. In someembodiments, the CD16 activating mutation is a mutation that results inhigher affinity to IgG1. In some embodiments, the CD16 comprises themutation 158V. In some embodiments, the CD16 comprises the mutation158F.

Also provided are engineered NK cells that have reduced surfaceexpression of an NK inhibitory receptor. In some embodiments, any of theprovided engineered NK cells that have reduced expression, activityand/or signaling of FcRγ chain can additionally have reduced surfaceexpression on an NK inhibitory receptor. In some embodiments, theengineered NK cells comprises a genetic disruption of a gene encoding aNK inhibitory receptor and/or a genetic disruption resulting in reducedexpression of an NK inhibitory receptor. In some embodiments, theengineered NK cell comprises an inhibitory nucleic acid that targets agene encoding a gene encoding an NK inhibitory receptor and/or resultsin reduced expression of an NK inhibitory receptor in the cell. In someembodiments, the inhibitory receptor is NKG2A and/or KIR2DL1. In someembodiments, expression of the inhibitory receptor, such as NKG2A and/orKIR2DL1, is reduced by greater than or greater than about 50%, 60%, 70%,80%, 90%, or 95% as compared to the expression, activity and/orsignaling in the NK cell that is not genetically engineered.

In some embodiments, the engineered NK cell exhibits increased activitywhen stimulated through CD16 compared to the NK cell that is notgenetically engineered. In some embodiments, the increased activity isobserved following CD16 engagement by CD16 crosslinking, such as canoccur in the presence of an antibody by binding of the Fc portion of theantibody to CD16.

In some embodiments, the engineered NK cell has reduced surfaceexpression of NKp46, NKp30, and/or NKp44 compared to the NK cell withoutthe modification. In some embodiments, expression of NKp46, NKp30,and/or NKp44 is reduced in the cell by greater than or greater thanabout 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared tothe expression in the NK cell that is not genetically engineered.

Also provided herein is a method of producing an engineered NK cell,comprising genetically engineering the NK cell to reduce FcRγ chainexpression, activity, and/or signaling in the cell. In some of theseembodiments, the methods involve disrupting a gene or repressingexpression a gene to effect reduced FcRγ chain expression, activityand/or signaling in the cell. In some cases, the genetic disruption canresult in a deletion or mutation of the gene. In some embodiments, themethods involves introducing into the cell an inhibitory nucleic acidthat reduces expression of a gene that results in reduced FcRγ chainexpression, activity and/or signaling in the cell.

In some embodiments, the method comprises disrupting a gene encodingFcRγ chain and/or disrupting a gene that results in reduced expressionof FcRγ chain in the engineered NK cell. In some embodiments, the methodcomprises disrupting a gene encoding a protein regulating expression oractivity of FcRγ chain and/or a disrupting a gene that results inreduced expression of a protein regulating expression or activity ofFcRγ chain. In some embodiments, the method comprises disrupting a geneencoding a protein involved in FcRγ chain-dependent signaling and/or adisrupting a gene that results in reduced expression of a proteininvolved in FcRγ chain-dependent signaling. In some embodiments, themethod of disrupting the gene results in introducing a deletion,mutation, or insertion into the gene.

In some embodiments, the method of disrupting the gene comprisesintroducing into an NK cell an endonuclease that has been engineered totarget to the gene. In some embodiments, the endonuclease is a TALnuclease, meganuclease, zinc-finger nuclease, CRISPR-associated protein9 (Cas9), or Argonaute. In some embodiments, the endonuclease is a Cas9that is a fusion with or is complexed with at least one guide RNA thatis complementary with a target domain or region of the gene. In someembodiments, the Cas9 is an S. aureus Cas9 molecule. In someembodiments, the Cas9 molecule is an S. pyogenes Cas9. In someembodiments, the method involves introducing into the cell anendonuclease, such as Cas9, that targets to the gene encoding FcRγ chainin the NK cell.

In some embodiments, the method for repressing expression of a genecomprises introducing an inhibitory nucleic acid into the cell thattargets a gene to result in reduced FcRγ chain expression, activityand/or signaling in the cell. In some embodiments, one or moreinhibitory nucleic acid molecule is introduced into the cell thattargets one or more genes resulting in reduced expression of a geneencoding FcRγ chain, a gene encoding a protein regulating expression oractivity of FcRγ chain and/or a gene encoding a protein involved in FcRγchain-dependent signaling. In some embodiments, the inhibitory nucleicacid comprises a sequence complementary to a gene gene encoding FcRγchain. In some embodiments, the inhibitory nucleic acid comprises asequence complementary to a gene encoding a protein regulatingexpression or activity of FcRγ chain. In some embodiments, theinhibitory nucleic acid comprises a sequence complementary to a geneencoding a protein involved in FcRγ chain-dependent signaling. In someembodiments, the inhibitory nucleic acid comprises an RNA interferingagent. In some embodiments, the nucleic acid is siRNA, shRNA, or miRNA.

In some of any such embodiments, the method is carried out to reduceexpression of a protein regulating expression or activity of FcRγ chainin the NK cell. In some of these embodiments, the protein regulatingexpression or activity of FcRγ chain is a transcription factor. In someembodiments, the transcription factor is PLZF (ZBTB16) or HELIOS(IKZF2).

In some of any such embodiments, the method is carried out to reduceexpression of a protein involved in FcRγ chain-dependent signaling inthe NK cell. In some embodiments, the protein involved in FcRγchain-dependent signaling is a downstream signaling molecule of FcRγ. Insome of these embodiments, the downstream signaling molecule is SYK,DAB2 or EAT-2.

In some of any such embodiments, the method is carried out to reduceexpression of FcRγ chain in the NK cell.

In some of any of the embodiments, the method is carried out in vitro.In some of any of the embodiments, the method is carried out ex vivo,such as from cells isolated from a subject, such as from a humanpatient. In some of any of the embodiments, the method is carried sothat the reduced expression is permanent, transient, or inducible.

In some embodiments, the methods for producing an engineered NK cellinvolves carrying out the genetic engineering, such as the disruption orrepression of expression of a gene as described, in primary NK cellsobtained from a subject. In some embodiments, the subject is mammalian,such as is human.

In some embodiments, prior to disrupting or repressing expression of thegene, the method comprises (i) isolating a NK cell from a sample from amammalian subject. In some embodiments, the mammalian subject is human.In some of these embodiments, the sample comprises peripheral bloodmononuclear cells (PBMC). In some embodiments, isolating the NK cellcomprises selecting NK cells based on surface expression of an NK cellmarker. In some embodiments, the NK cell marker is one or more of CD56,CD161, KIR, NKG2A, NKG2D, NKp30, NKp44, NKp46, 2B4, NTB-A, CRACC,DNAM-1, CD69, and/or CD25. In some embodiments, the method comprisesselecting NK cells from other lymphocytes by selecting cells that do notexpress surface CD3, a T-cell antigen receptor (TCR) and/or surfaceimmunoglobulins (Ig) B cell receptor. In some embodiments, the NK cellsare selected or are additionally selected to have surface expression ofCD16. In some embodiments, the NK cells express CD3ζ.

In some embodiments, the methods for producing an engineered NK cellinvolves carrying out the genetic engineering on an NK cell line. Insome embodiments, the cell line is NK-92, NK-YS, KHYG-1. NKL, NKG,SNK-6, or IMC-1.

In some embodiments, the method comprises engineering the NK cell lineto express a recombinant or heterologous CD16 and/or CD3ζ. In someembodiments, the method comprises introducing into the NK cell a nucleicacid encoding the CD16 and/or CD3ζ. In some embodiments, the recombinantor heterologous CD16 contains an activating mutation. In someembodiments, the activating mutation increases affinity of CD16 for IgG.In some embodiments, the CD16 comprises a 158V mutation. In someembodiments, the CD16 comprises a 158F mutation.

In some embodiments, the method comprises virally transducing the NKcell with nucleic acid encoding the CD16 and/or CD3ζ gene. In someembodiments, the method comprises transfecting the NK cell with nucleicacid encoding the CD16 and/or CD3ζ gene. In some embodiments, the methodcomprises transiently, inducibly, or permanently expressing CD16 or CD3ζin the NK cell.

In some embodiments, the method is carried out so that FcRγ chainexpression, signaling and/or activity is reduced in the NK cell bygreater than or greater than about 50%, 60%, 70%, 80%, 90%, or 95% ascompared to the expression in the NK cell that is not geneticallyengineered by the present method. In some embodiments, the FcRIγ adaptorprotein expression level is not detectable by an immunoblot assay.

Also provided is a method of producing an engineered NK cell that hasreduced surface expression of an NK inhibitory receptor. In someembodiments, any of the provided methods for producing an engineered NKcell that have reduced expression, activity and/or signaling of FcRγchain can additionally involve reducing surface expression on an NKinhibitory receptor. In some embodiments, such engineered NK cells areproduced by a method that includes disrupting a gene encoding a NKinhibitory receptor. In some embodiments, such engineered NK cells areproducing by introducing into the NK cell an inhibitory nucleic acidthat targets a gene encoding an NK inhibitory receptor. In someembodiments, the inhibitory receptor is NKG2A and/or KIR2DL1. In someembodiments, such methods are carried out so that expression of an NKinhibitory receptor, such as NKG2A and/or KIR2DL1, is reduced in the NKcell by greater than or greater than about 50%, 60%, 70%, 80%, 90%, or95% as compared to the expression of the inhibitory receptor in the NKcell that is not genetically engineered by the present method.

In some embodiments, the provided methods for producing geneticallyengineered NK cell can further include a step of expanding theengineered NK cells. In some embodiments, expanding the engineered NKcells involves culturing or incubating the engineered NK cells in thepresence of feeder cells or cytokines. In some embodiments, expandingthe engineered NK cells in carried out in vitro. In some embodiments,expanding the engineered NK cells is carried out in vivo.

Also provided are engineered NK cells produced by any of the abovemethods.

Also provided herein are compositions comprising any of the providedengineered NK cells. In one embodiment, the composition comprises atherapeutically effective amount of the engineered NK cells providedherein and a pharmaceutically acceptable carrier. In some embodiments,the carrier is a saline solution, a dextrose solution, or 5% human serumalbumin. In some embodiments, the composition comprises between 1×10⁵and 1×10⁸ cells/mL.

In some embodiments, the composition is a cryopreserved compositionand/or comprises the engineered NK cells and a cryoprotectant.

Also provided here are kits comprising an engineered NK cell and anadditional agent. In some embodiments, the kits further compriseinstructions for use, for example, instructions for administering theengineered NK cell and additional agent for treatment of a disease. Insome embodiments, the additional agent is an antibody or an Fc-fusionprotein. In some embodiments, the antibody recognizes a tumor associatedantigen, a viral antigen, a microbial antigen. In some embodiments, theantibody is selected from the group consisting of an anti-CD20 antibody,an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody andan anti-CD38 antibody. In some embodiments, the antibody comprises an Fcdomain.

Also provided herein is a method of treating a condition comprisingadministering any of the provided engineered NK cells to an individualin need thereof. In some embodiments, the individual is a mammaliansubject, such as a human subject. In some embodiments, the subject isone that has a cancer or an infection, such as a viral infection ormicrobial infection. In some embodiments, the method comprisesadministering 1×10⁸ to 1×10¹⁰ cells/m² to the individual.

In some embodiments, the provided methods further include administeringan additional agent. In some embodiments, the additional agent is anantibody or an Fc-fusion protein. In some embodiments, the antibodyrecognizes a tumor associated antigen, a viral antigen, a microbialantigen. In some embodiments, the antibody is an anti-CD20 antibody, ananti-HER2 antibody, an anti-CD52 antibody, an anti-EGFR antibody or ananti-CD38 antibody. In some embodiments, the antibody comprises an Fcdomain.

In some embodiments, the additional agent and the engineered NK cellsare administered sequentially. In some embodiments, the additional agentis administered prior to administration of the engineered NK cells. Insome embodiments, the additional agent and the engineered NK cells areadministered simultaneously.

In some embodiments, the method comprises administering NK cells totreat an inflammatory condition, an infection, and/or cancer. In someembodiments, the infection is a viral infection or a bacterialinfection. In some embodiments, the cancer is leukemia or lymphoma. Insome embodiments, the individual expresses a low affinity FcγRIIIA. Insome embodiments, the individual is a human.

In some embodiments, the engineered NK cell is allogenic to the subject.In some embodiments, the engineered NK cell is autologous to thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts activity of g-NK cells and conventional NK cells in anantibody dependent cell cytotoxicity (ADCC) assay in the absence orpresence of anti-CD20 antibody Rituximab.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are engineered Natural Killer (NK) cells that aregenetically engineered to have reduced expression, activity and/orsignaling of the FcRγ (also known as FcεRIγ). In some aspects, theprovided engineered NK cells are genetically engineered to knockout(e.g. by genetic disruption) or knockdown (e.g. by gene silencing orrepression) a gene encoding FcRγ, a gene encoding a protein controllingexpression of the FcRγ, such as a transcription factor, or a geneencoding a protein involved in FcRγ-dependent signaling, such as adownstream signaling molecule of FcRγ.

Natural killer (NK) cells are innate lymphocytes important for mediatinganti-viral and anti-cancer immunity through cytokine and chemokinesecretion, and through the release of cytotoxic granules (Vivier et al.Science 331(6013):44-49 (2011); Caligiuri, Blood 112(3):461-469 (2008);Roda et al., Cancer Res. 66(1):517-526 (2006)). NK cells are effectorcells that comprise the third largest population of lymphocytes and areimportant for host immuno-surveillance against tumor andpathogen-infected cells.

However, unlike T and B lymphocytes, N K cells are thought to have onlya limited capacity for target recognition using germline-encodedactivation receptors (Bottino et al., Curr Top Microbiol Immunol.298:175-182 (2006); Stewart et al., Curr Top Microbiol Immunol. 298:1-21(2006)). Instead, NK cells express the activating Fc receptor CD16,which recognizes IgG-coated target cells, thereby broadening targetrecognition (Ravetch & Bolland, Annu Rev Immunol. 19:275-290 (2001);Lanier Nat. Immunol. 9(5):495-502 (2008); Bryceson & Long. Curr OpinImmunol. 20(3):344-352 (2008)). In some cases, antibody-dependentcellular cytotoxicity (ADCC) is triggered when receptors on the NK cellsurface (such as CD16) recognize IgG1 or IgG3 antibodies bound to thesurface of a cell. This triggers release of cytoplasmic granulescontaining perforin and granzymes, leading to target cell death. ADCCand antibody-dependent cytokine/chemokine production are primarilymediated by NK cells.

In some cases, ADCC is a mechanism of action of therapeutic antibodies,including anti-cancer antibodies. While significant advances have beenmade in cancer treatment by use of antibodies directed against cancerantigens, the responsiveness of patients to such antibodies varies.Investigation of such variable responses has typically focused on thedirect inhibitory effects of these antibodies on the tumor cells (e.g.inhibition of growth factor receptors and the subsequent induction ofapoptosis) and the in vivo effects of these antibodies may be morecomplex and may involve the host immune system, including through ADCC.In some aspects, cell therapy by administering NK cells can be used inconcert with antibodies for therapeutic and related purposes.

Upon activation. NK cells produce cytokines and chemokines abundantlyand at the same time exhibit potent cytolytic activity. Activation of NKcells can occur through the direct binding of NK cell receptors toligands on the target cell, as seen with direct tumor cell killing, orthrough the crosslinking of the Fc receptor (CD 16; FcγRIII) by bindingto the Fc portion of antibodies bound to an antigen-bearing cell. Theexpression and signal transduction activity of several NK cellactivation receptors requires physically associated adaptors, whichtransduce signals through immunoreceptor tyrosine-based activationmotifs (ITAMs). Among these adaptors. FcRγ and CD3ζ chains can associatewith CD16 and natural cytotoxicity receptors (NCRs) as eitherdisulfide-linked homo-dimers or hetero-dimers, and these chains havebeen thought to be expressed by all mature NK cells.

In some aspects, CD16 engagement (CD16 crosslinking) initiates NK cellresponses via intracellular signals that are generated through one, orboth, of the CD16-associated adaptor chains, FcRγ or CD3ζ. Triggering ofCD16 leads to phosphorylation of the γ or ζ chain, which in turnrecruits tyrosine kinases, syk and ZAP-70, initiating a cascade ofsignal transduction leading to rapid and potent effector functions. Themost well-known effector function is the release of cytoplasmic granulescarrying toxic proteins to kill nearby target cells through the processof antibody-dependent cellular cytotoxicity. CD16 crosslinking alsoresults in the production of cytokines and chemokines that, in turn,activate and orchestrate a series of immune responses.

This release of cytokines and chemokines can play a role in theanti-cancer activity of NK cells in vivo. NK cells also have smallgranules in their cytoplasm containing perforin and proteases(granzymes). Upon release from the NK cell, perforin forms pores in thecell membrane of targeted cells through which the granzymes andassociated molecules can enter, inducing apoptosis. The fact that NKcells induce apoptosis rather than necrosis of target cells issignificant-necrosis of a virus-infected cell would release the virions,whereas apoptosis leads to destruction of the virus inside the cells.

The provided engineered NK cells include cells that are engineered tohave lower activity or expression of FcRγ signaling adaptor. In somecases, the engineered cells express the signaling adaptor CD3ζ-chainabundantly, but are deficient in the expression of the signaling adaptorFcRγ-chain. In some embodiments, compared to NK cells that express thesignaling adaptor FcRγ-chain, these engineered NK cells exhibitdramatically enhanced activity when activated by engagement of CD16,such as can occur in the presence of antibodies. For example, theengineered cells can be activated by antibody-mediated crosslinking ofCD16 or by antibody-coated tumor cells. Also provided are methods ofproducing the engineered cells. The provided engineered NK cells andmethods address problems related to selecting or identifying NK cells ofa particular phenotype, which may only be present as a small percentageof total NK cells in a subject and/or which may not normally exist inall subjects in a population. In some aspects, the provided embodimentsoffer an improved NK cell therapy in which NK cells can be engineeredwith enhanced activities and yet can be more easily obtained insufficient amounts for therapeutic use, including for administration inconcert with antibodies, compared to NK cells of a similar phenotypeisolated directly from a subject.

All references cited herein, including patent applications, patentpublications, and scientific literature and databases, are hereinincorporated by reference in their entirety for all purposes to the sameextent as if each individual reference were specifically andindividually indicated to be incorporated by reference.

For clarity of disclosure, and not by way of limitation, the detaileddescription is divided into the subsections that follow. The sectionheadings used herein are for organizational purposes only and are not tobe construed as limiting the subject matter described.

I. Definitions

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, “antibody” refers to immunoglobulins and immunoglobulinfragments, whether natural or partially or wholly synthetically, such asrecombinantly, produced, including any fragment thereof containing atleast a portion of the variable heavy chain and/or light chain region ofthe immunoglobulin molecule that is sufficient to form an antigenbinding site and, when assembled, to specifically bind antigen. Hence,an antibody includes any protein having a binding domain that ishomologous or substantially homologous to an immunoglobulinantigen-binding domain (antibody combining site). Typically, antibodiesminimally include all or at least a portion of the variable heavy(V_(H)) chain and/or the variable light (V_(L)) chain. In general, thepairing of a V_(H) and V_(L) together form the antigen-binding site,although, in some cases, a single V_(H) or V_(L) domain is sufficientfor antigen-binding. The antibody also can include all or a portion ofthe constant region. Reference to an antibody herein includesfull-length antibody and antigen-binding fragments. The term“immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antibody fragment. A full-length antibodyis an antibody typically having two full-length heavy chains (e.g.,VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains(VL-CL) and hinge regions, such as antibodies produced from mammalianspecies (e.g. human, mouse, rat, rabbit, non-human primate, etc.) byantibody secreting B cells and antibodies with the same domains that areproduced synthetically. Specifically whole antibodies include those withheavy and light chains including an Fc region. The constant domains maybe native sequence constant domains (e.g., human native sequenceconstant domains) or amino acid sequence variants thereof. In somecases, the intact antibody may have one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody, theantigen binding and/or the variable region of the intact antibody.Antibody fragments, include, but are not limited to, Fab fragments, Fab′fragments, F(ab′)₂ fragments. Fv fragments, disulfide-linked Fvs (dsFv),Fd fragments. Fd′ fragments; diabodies: linear antibodies (see U.S. Pat.No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062[1995]); single-chain antibody molecules, including single-chain Fvs(scFv) or single-chain Fabs (scFab); antigen-binding fragments of any ofthe above and multispecific antibodies from from antibody fragments. Forpurposes herein, an antibody fragment typically includes one that issufficient to engage or crosslink CD16 on the surface of an NK cell.

The term “autologous” refers to cells or tissues originating within ortaken from an individual's own tissues. For example, in an autologoustransfer or transplantation of NK cells, the donor and recipient are thesame person.

The term “allogeneic” refers to cells or tissues that belong to or areobtained from the same species but that are genetically different, andwhich, in some cases, are therefore immunologically incompatible.Typically, the term “allogeneic” is used to define cells that aretransplanted from a donor to a recipient of the same species.

The term “expression” refers to the process by which a polynucleotide istranscribed from a DNA template (such as into an mRNA or other RNAtranscript) and/or the process by which a transcribed mRNA issubsequently translated into peptide, polypeptides or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

The term “heterologous” with reference to a protein or nucleic acidrefers to a protein or nucleic acid originating from a different geneticsource. For example, a protein or nucleic acid that is heterologous to acell originates from an organism or individual other than the cell inwhich it is expressed.

As used herein, the term “introducing” encompasses a variety of methodsof introducing DNA into a cell, either in vitro or in vivo, such methodsincluding transformation, transduction, transfection (e.g.electroporation), and infection. Vectors are useful for introducing DNAencoding molecules into cells. Possible vectors include plasmid vectorsand viral vectors. Viral vectors include retroviral vectors, lentiviralvectors, or other vectors such as adenoviral vectors or adeno-associatedvectors.

The term “composition” refers to any mixture of two or more products,substances, or compounds, including cells or antibodies. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof. The preparation is generally in such form as topermit the biological activity of the active ingredient (e.g. antibody)to be effective.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, combination refers to any association between or amongtwo or more items. The combination can be two or more separate items,such as two compositions or two collections, can be a mixture thereof,such as a single mixture of the two or more items, or any variationthereof. The elements of a combination are generally functionallyassociated or related.

As used herein, a kit is a packaged combination that optionally includesother elements, such as additional agents and instructions for use ofthe combination or elements thereof, for a purpose including, but notlimited to, therapeutic uses.

As used herein, the term “treatment” or “treating” refers to clinicalintervention designed to alter the natural course of the individual orcell being treated during the course of clinical pathology. Desirableeffects of treatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. An individual is successfully “treated”, for example, if oneor more symptoms associated with a disorder (e.g., aneosinophil-mediated disease) are mitigated or eliminated. For example,an individual is successfully “treated” if treatment results inincreasing the quality of life of those suffering from a disease,decreasing the dose of other medications required for treating thedisease, reducing the frequency of recurrence of the disease, lesseningseverity of the disease, delaying the development or progression of thedisease, and/or prolonging survival of individuals.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired or indicatedeffect, including a therapeutic or prophylactic result. An effectiveamount can be provided in one or more administrations. A“therapeutically effective amount” is at least the minimum dose of cellsrequired to effect a measurable improvement of a particular disorder. Atherapeutically effective amount herein may vary according to factorssuch as the disease state, age, sex, and weight of the patient. Atherapeutically effective amount may also be one in which any toxic ordetrimental effects of the antibody are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at the dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at the earlier stage of disease, the prophylactically effectiveamount can be less than the therapeutically effective amount.

As used herein, an “individual” or a “subject” is a mammal. A “mammal”for purposes of treatment includes humans, domestic and farm animals,and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle,pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In someembodiments, the individual or subject is human.

II. Engineered NK Cells

Provided herein is an engineered NK cell that is genetically engineeredto reduce FcRγ chain expression, activity and/or signaling in the cell.Also provided are methods for engineering the cells. In someembodiments, the methods include introducing a genetic disruption orinhibitory nucleic acid that disrupts a gene or represses expression ofa gene resulting in reduced expression, activity and/or signaling ofFcRγ chain in the NK cell.

In some embodiments, the engineered NK cell is genetically engineered todirectly reduce or eliminate expression or activity of FcRγ chain. Insome embodiments, the engineered NK cell is genetically engineered toindirectly reduce or eliminate expression or activity of FcRγ chain,such as by reducing or eliminating expression of a protein thatregulates expression or activity of FcRγ, for example a transcriptionfactor that controls expression of FcRγ. In some embodiments, theengineered NK cell is genetically engineered to reduce or eliminateexpression or activity of a molecule involved in the downstreamsignaling of FcRγ. In some aspects, the engineered cell retains or isadditionally engineered to transduce signals through CD3ζ, such as uponengagement or crosslinking of CD16.

Targets for Engineering

In some embodiments the NK cell is genetically engineered to reduce oreliminate expression or activity of human FcRγ chain protein. In someembodiments, the engineered NK cell comprises a genetic disruption of agene encoding FcRγ chain or of a gene encoding a protein that regulatesexpression or activity of FcRγ chain protein. In some embodiments, thegenetic disruption results in an insertion, deletion or mutation in thegene, such as a frameshift mutations and/or premature stop codons withinthe open reading frame. In some embodiments, one allele is disrupted. Insome embodiments, both alleles are disrupted. In some embodiments theengineered NK cell comprises an inhibitory nucleic acid, such as ansiRNA or other inhibitory nucleic acid molecule, that reduces expressionof a gene encoding FcRγ chain or of a gene encoding a protein thatregulates expression or activity of FcRγ chain protein.

An amino acid sequence for FcRγ chain (Homo sapiens, also called theHigh affinity immunoglobulin gamma Fc receptor I) is available in theNCBI database as accession number NP_004097.1 (GI:4758344), and isreproduced below as SEQ ID NO:1.

 1 MIPAVVLLLL LLVEQAAALG EPQLCYILDA ILFLYGPVLT 41LLYCRLKIQV RKAAITSYEK SDGVYTGLST RNQETYETLK 81 HEKPPQ

The NCBI database genomic reference sequence for FcRγ chain isNG_029043.1 RefSeqGene. The mRNA reference sequence is 1.NM_004106.1.

The engineered N K cell may contain a mutation, disruption, or deletionin the gene encoding FcRγ signaling adaptor, for example an inactivatingmutation in an exon of the FcRγ chain gene. The mutation, disruption, ordeletion can also be in a regulatory element of the FcRγ chain gene, forexample, in the promoter.

Various inactivating mutations or deletions can also be used to decreaseexpression of FcRγ chain. For example, a mutation in the immunoreceptortyrosine-based activation motif (ITAM) may be used. In some embodiments,the engineered NK cells may comprise an inactivating mutation in theITAM motif. In other embodiments, the transmembrane region of FcRγ chainmay be mutated to inactivate the protein.

In some embodiments, it is also possible to decrease FcRγ chainexpression or activity, indirectly by increasing or decreasingexpression of a protein that regulates FcRγ chain. In some embodiments,the engineered NK cell is genetically engineered to reduce or eliminateexpression of a protein that regulates expression or activity of FcRγchain, such as a transcription factor, e.g. PLZF or HELIOS. In someembodiments, the engineered NK cell comprises a genetic disruption of agene encoding a protein that regulates expression or activity of FcRγchain protein. In some embodiments, the genetic disruption results in aninsertion, deletion or mutation in the gene, such as a frameshiftmutations and/or premature stop codons within the open reading frame. Insome embodiments, one allele is disrupted. In some embodiments, bothalleles are disrupted. In some embodiments the engineered NK cellcomprises an inhibitory nucleic acid that reduces expression of a geneencoding a protein that regulates expression or activity of FcRγ chainprotein, such as a transcription factor, e.g. PLZF or HELIOS.

In some embodiments, the engineered NK cell may contain a geneticdisruption, deletion, or mutation in a gene that promotes transcriptionof FcRγ signaling adaptor. In some embodiments, the engineered NK cellmay have a genetic disruption, mutation, or deletion in a gene encodinga transcription factor that promotes transcription of the FcRγ chain.For example, the engineered NK cell may have a genetic disruption, in agene encoding the PLZF or HELIOS transcription factor. In someembodiments, one allele of PLZF and/or HELIOS is disrupted. In someembodiments, both alleles of PLZF or both alleles of HELIOS aredisrupted. In some embodiments, the engineered NK cell contains aninhibitory nucleic acid molecule, such as an siRNA or other inhibitorynucleic acid molecule, that reduces expression of a gene that promotestranscription of FcRγ signaling adaptor. In some embodiments, theengineered NK cell may have an inhibitory nucleic acid molecule thattargets a transcription factor that promotes transcription of the FcRγchain, such as an inhibitory nucleic acid molecule that targets a geneencoding the PLZF or HELIOS transcription factor.

The amino acid sequence of PLZF is available in the NCBI database asaccession number NP_001018011.1, and is reproduced below as SEQ ID NO:3.The NCBI database genomic reference sequence is NG_012140.1. The mRNAreference sequence is NM_001018011.1.

MDLTKMGMIQLQNPSHPTGLLCKANQMRLAGTLCDVVIMVDSQEFHAHRTVLACTSKMFEILFHRNSQHYTLDFLSPKTFQQILEYAYTATLQAKAEDLDDLLYAAEILEIEYLEEQCLKMLETIQASDDNDTEATMADGGAEEEEDRKARYLKNIFISHSSEESGYASVAGQSLPGPMVDQSPSVSTSFGLSAMSPTKAAVDSLMTIGQSLLQGTLQPPAGPEEPTLAGGGRHPGVAEVKTEMMQVDEVPSQDSPGAAESSISGGMGDKVEERGKEGPGTPTRSSVITSARELHYGREESAEQVPPPAEAGQAPTGRPEHPAPPPEKHLGIYSVLPNHKADAVLSMPSSVTSGLHVQPALAVSMDFSTYGGLLPQGFIQRELFSKLGELAVGMKSESRTIGEQCSVCGVELPDNEAVEQHRKLHSGMKTYGCELCGKRFLDSLRLRMHLLAHSAGAKAFVCDQCGAQFSKEDALETHERQTHTGTDMAVFCLLCGKRFQAQSALQQHMEVHAGVRSYICSECNRTFPSHTALKRHLRSHTGDHPYECEFCGSCFRDESTLKSHKRIHTGEKPYECNGCGKKFSLKHQLETHYRVHTGEKPFECKLCHQRSRDYSAMIKHLRTHNGASPYQCTICTEYCPSLSSMQKHMKGHKPEEIPPDWRIEKTYLYLCYV

The amino acid sequence of HELIOS (IKZF2) is available in the NCBIdatabase as accession number NP_057344.2 (isoform 1) and NP_001072994.1(isoform 2) The NCBI gene identifier is NC_000002.12. The mRNA referencesequence is NM_016260.2 (isoform 1) NM_001079526.1 (isoform 2). The geneID is 22807.

In some embodiments, the engineered NK cell is genetically engineered toreduce or eliminate a protein involved in FcRγ chain-dependentsignaling. In some embodiments, the engineered NK cell comprises areduction in FcRγ chain-dependent signaling. For example, the engineeredNK cell may have reduced expression of downstream molecules that isinvolved in FcRγ chain-dependent signaling. For example. Lee and Schlumsdescribe members of the FcRγ signaling pathway that can be suitabletargets for modification and/or deletion (Lee et al., Immunity 42:431-42(2015); Schlums et al. Immunity, 42:443-56 (2015)). In some of theseembodiments, the downstream molecules comprise SYK. DAB2 or EAT2. Thegene ID for SYK is 6850. The NCBI gene ID for DAB2 is 1601. The genomicreference sequence for DAB2 is NG_030312.1. The mRNA and proteinsequences for isoform 2 of DAB2 are 001244871.1 and NP_001231800.1. ThemRNA and protein sequences for isoform 1 of DAB2 are 2.NM_001343.3 andNP_001334.2. The NCBI gene ID for EAT2 is 175072.

In some embodiments, the engineered NK cell may contain a have a geneticdisruption, mutation, or deletion in a gene encoding a protein involvedin FcRγ chain-dependent signaling. For example, the engineered NK cellmay have a genetic disruption, in a gene encoding the SYK, DAB2 and/orEAT2. In some embodiments, one allele of SYK. DAB2 and/or EAT2 isdisrupted. In some embodiments, both alleles of SYK, both alleles ofDAB2 and/or both alleles of EAT2 are disrupted. In some embodiments, theengineered NK cell contains an inhibitory nucleic acid molecule, such asan siRNA or other inhibitory nucleic acid molecule, that reducesexpression of a gene that is involved in FcRγ chain-dependent signaling.In some embodiments, the engineered NK cell may have an inhibitorynucleic acid molecule that targets a signaling molecule, such as aninhibitory nucleic acid molecule that targets a gene encoding SYK, DAB2and/or EAT2.

Also provided are engineered NK cells that are genetically engineered toreduce or eliminate expression of an NK inhibitory receptor. In someaspects, any of the provided engineered that are engineered to havedecreased expression, signaling, and/or activity of FcRγ chain asdescribed above additionally can be engineered to reduce or eliminateexpression of an NK inhibitory receptor. In some cases, the engineeredcell contains a disruption of a gene encoding the NK inhibitoryreceptor. In some embodiments, the engineered cell contains aninhibitory nucleic acid molecule, such as an siRNA or other inhibitorynucleic acid molecule, that reduces expression of a gene encoding the NKinhibitory receptor. Inhibitory NK receptors are known in the art andinclude NKG2A (also known as KLRC1; NCBI gene ID 3821) and KIR2DL1 (NCBIgene ID 3802). The NK cell may be engineered to decrease expression ofthe inhibitory NK receptor using any of the methods provided herein, forexample using an interfering RNA or genetic disruption, including a geneinsertion, mutation, or deletion that produces a stop codon orframeshift.

In the above embodiments, such engineered NK cells include those thatcomprise genetic disruptions produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce non-conservative amino acid substitutions, deletions oradditions. In some embodiments, the genetic disruption comprises aninsertion, deletion, or mutation that results in a premature stop codonin the gene or a frameshift of the open reading frame of the gene.

In some embodiments, the expression of a particular gene product, suchas any described above, in the engineered cell is reduced by greaterthan about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,98% or 99% as compared to the expression of the gene product in the NKcell that is not genetically engineered. In some embodiments, theexpression of a particular gene product in the engineered cell isreduced to a level that is undetectable. In some embodiments, theexpression of a particular gene product in the engineered cell iscompletely eliminated. In some of these embodiments, the gene is FcRγchain. In other embodiments, the gene may encode a protein thatregulates FcRγ chain, such as a transcription factor that promotesexpression of FcRγ chain, such as PLZF or HELIOS. In other embodiments,the gene may encode a protein that is involved in FcRγ chain-mediatedsignaling, such as a such as downstream signaling molecules SYK, DAB2 orEAT2.

In some embodiments, the expression, activity and/or signaling of FcRγchain in the provided engineered NK cells is reduced by greater than10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%as compared to the expression of the gene in the NK cell that is notgenetically engineered. In some of these embodiments, the level of FcRγchain expression is reduced to an undetectable level using an immunoblotassay.

In some embodiments, the engineered NK cell may express CD16 (alsoreferred to as CD16A or FcyRIIIa). Thus, reference to CD16 herein is notmeant to refer to the glycosylphosphatidylinositol-anchored form(FcγRIIIB or CD16B). In some of these embodiments, the CD16 is humanCD16. In humans or as a glycosylphosphatidylinositol-anchored form(FcγRIIIB or CD16B). Typically, the provided engineered cells express apolypeptide-anchored CD16 form that is able to associated with the ζchain of the TCR-CD3 complex. CD16 binds to antibody Fc regions andinitiates ADCC. In some embodiments, the expression of other Fe receptorproteins is maintained in the engineered NK cell.

The genomic sequence for CD16A is available in the NCBI database atNG_009066.1. The gene ID for CD16A is 2214. Sequence information forCD16, including gene polymorphisms, is available at UniProt Ace. No.P08637. Nucleic acid and protein sequences for CD16a are publiclyavailable. For example, GenBank Accession Nos. NM_000569 (SEQ ID NO: 1),NM_001127596. NM_001127595, NM_001127593, and NM_001127592 discloseexemplary human CD16a nucleic acid sequences, and GenBank Accession Nos.NP_000560 (SEQ ID NO: 2), NP_001121068, NP_001121067, NP_00112065, andNP_001121064 disclose exemplary human CD16a protein sequences. One ofordinary skill in the art can identify additional CD16a nucleic acid andamino acid sequences that vary from those provided herein, but thatretain at least one activity of CD16a, such as Fc binding activity.

CD16 is most commonly found in a form that has a relatively low bindingaffinity for the Fc portion of IgG molecules. An alternative form thatexhibits a higher binding affinity is found in some individuals. The lowand high affinity forms of CD16 differ only by the substitution ofvaline (high affinity) for phenylalanine (low affinity) at position 158in the mature (processed) form of the polypeptide chain. The sequence ofCD16 (158F) is set forth in SEQ ID NO:4 (residue 158F is bold andunderlined). In some embodiments, CD16 (158F) further comprises a signalpeptide set forth as MWQLLLPTALLLLVSA (SEQ ID NO:5).

(SEQ ID NO: 4) GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKD SGSYFCRGL FGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

The sequence of CD16 158V (polymorphism resulting in F158V) is known asVAR_003960 and has the sequence set forth in SEQ ID NO:6 (158Vpolymorphism is in bold and underline). In some embodiments, CD16 (158V)further comprises a signal peptide set forth as MWQLLLPTALLLLVSA (SEQ IDNO:5).

(SEQ ID NO: 6) GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKD SGSYFCRGL VGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

In some embodiments, the engineered NK cell may comprise a CD16 genethat comprises an activating mutation. In some of embodiments, themutation results in higher affinity to an IgG region. In some of theseembodiments, the CD16 mutation results in higher affinity of CD16 forIgG1, IgG2, or IgG4. In some embodiments, the CD16 mutation results inhigher affinity of CD16 for IgG1. In some embodiments, the CD16 containsthe 158V mutation. In some embodiments, the CD16 has the sequence ofamino acids set forth in SEQ ID NO:6 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%,93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6 and thatcontains the 158V polymorphism. In some embodiments, the mutation inCD16 is a 158F mutation. In some embodiments, the CD16 has the sequenceof amino acids set forth in SEQ ID NO:4 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%,93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 andthat contains the 158F polymorphism.

In some aspects, the engineered NK cells can be engineered to express arecombinant or heterologous CD16. In some embodiments, even if the NKcells may express CD16, the NK cells may be engineered to furtherexpress a higher level of CD16 and/or express a modified form of CD16than is expressed by the NK in the absence of the genetic engineering.In such examples, the cells can be engineered to contain nucleic acidcomprising a strong or constitutive promotor followed by a CD16 gene.

The engineered NK cell may also express other signaling adaptormolecules. For example, signaling molecules with an ITAM domain otherthan FcRγ. In some embodiments, the engineered NK cell expresses CD3ζadaptor chain. In some of these embodiments, CD3ζ is human CD3ζ.

An amino acid sequence for the human CD3ζ (Homo sapiens) is available inthe NCBI database as accession number ABQ28690.1 (GI: 146399947), and isreproduced below as SEQ ID NO:2.

 1 AILQAQLPIT EAQSFGLLDP KLCYLLDGIL FIYGVILTAL 41FLRVKFSRSA DAPAYQQGQN QLYNELNLGR REEYDVLDKR 81 RG

The NCBI gene ID for CD3ζ (CD247) is 919.

In some aspects, the engineered NK cells can be engineered to express arecombinant or heterologous CD3ζ. In some embodiments, even if the NKcells may express CD3ζ, the NK cells may be engineered to furtherexpress a higher level of CD3ζ and/or express a modified form of CD3ζthan is expressed by the NK in the absence of the genetic engineering.In such examples, the cells can be engineered to contain nucleic acidcomprising a strong or constitutive promotor followed by a CD3ζ gene. Insome embodiments, the engineered cells may comprise a CD3ζ gene with anactivating mutation. In other embodiments, the engineered NK cells mayexpress CD3ζ at a higher level after engineering.

In some embodiments, the engineered NK cell may have reduced expressionof NK cell surface receptors, including natural cytotoxicity receptors.The engineered NK cell may have reduced expression of NKp46, NKp30,and/or NKp44 compared to the NK cell without the modification. In someof these embodiments, the expression of the cell surface receptor isreduced by greater than 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 10fold, 100 fold or 1000 fold. In some of these embodiments the NK cellsurface receptor may not be detectable in the engineered NK cell. Inother embodiments, the NK cell may be negative for NK cell surfacereceptors, such as NKp46, NKp30, and/or NKp44.

Cell Types

One of skill in the art will appreciate that both primary cells obtainedfrom human tissue and existing cell lines are suitable for engineering.For example, in some embodiments, the engineered NK cell may be derivedfrom a primary cell obtained from a subject, such as a human.

Primary Cells

Primary cells can be engineered using any of the methods providedherein. According to some embodiments the population of cells comprisingsaid NK cells is obtained from a sample from a mammalian subject, suchas a human subject. The sample or source can be cord blood, bone marrowor peripheral blood.

In some aspects, Natural killer cells expressing one or more naturalkiller cell-specific markers are isolated from the cell population.Methods of isolating and identifying NK cells are well known in the art,and include those discussed in Dahlberg et al, Frontiers in Immunology,vol. 6 article 605 pp. 1-18 (2015).

Techniques for the in vitro isolation and large-scale expansion of NKcells are known. An exemplary procedure is described in US Pat. App.Publ. No. 2014/0086890, incorporated herein by reference in itsentirety. One of ordinary skill in the art can identify additionalmethods for expanding NK cells, for example as described in Childs etal., Hematol. The Education Program 2013:234-246, 2013, incorporatedherein by reference in its entirety.

In some embodiments, mononuclear cells are collected from a subject(such as a donor subject or a subject with a tumor or hyperproliferativedisease). In some examples, mononuclear cells are collected by anapheresis procedure. The mononuclear cells are enriched for NK cells,for example by negative depletion using an immuno-magnetic beadstrategy. In some examples, NK cells are enriched by depleting themononuclear cell sample of T cells, B cells, monocytes, dendritic cells,platelets, macrophages, and erythrocytes utilizing a mixture ofbiotinylated monoclonal antibodies. The non-NK cells in the sample areremoved with magnetic beads coupled to streptavidin, resulting in anenriched preparation of NK cells. An exemplary commercially availablekit for this method is Dynabeads® Untouched™ Human NK Cells kit (ThermoFisher Scientific, Waltham, Mass.). In another example, NK cells areenriched by positive selection of CD56′ NK cells, for example utilizingmagnetic beads conjugated to an anti-CD56 antibody (such as CD56MicroBeads, Miltenyi Biotec, Inc., Auburn, Calif.). In other examples, atwo-step method including negative depletion (such as T cell depletion)followed by positive selection of CD56⁺ NK cells is used for enrichingNK cells.

In some embodiments, Natural killer cells may be identified as thoseexpressing typical human natural killer cell markers such as KIR, NKG2A,NKG2D, NKp30, NKp44, NKp46, CD56, and CD161. For studies involving mice,natural killer cells can be identified and/or isolated using typicalmouse markers such as NKI.1, CD 122, LY49 Family (Ly49A, Ly49C, Ly49D,Ly49E, Ly49F, Ly49G, Ly49H, and Ly49I), or NKG2A/C/E. In someembodiments, cell staining, or FACS may be used to identify cells thatexpress a certain marker.

In some embodiments, NK cells can be selectively enriched using eitherpositive or negative selection. For example, NK cells that do notexpress CD3 can be selected by exposing a mixture of cells to animmobilized anti-CD3 antibody and removing the unbound cells. Accordingto some embodiments of the present invention the NK cells compriseCD56+CD3− cells. According to some embodiments of the present inventionthe NK cells comprise CD56+CD16+CD3− cells.

In one embodiment, cytokines can be administered to a subject prior toisolating primary NK cells. For example, IL-12, IL-15, IL-18, IL-2,and/or CCL5 can be administered to a subject prior to isolating theprimary NK cells.

It may also be beneficial to enrich the isolated NK primary cells forthose that express CD16 and CD3ζ. One of skill in the art willappreciate that many methods exist for selectively enriching cellsexpressing certain markers from population exist, such as fluorescentcell sorting, or selective depletion of cells expressing certain markersusing antibodies bound to a solid phase.

In some embodiments, the methods can be carried out under or adapted forCurrent Good Manufacturing Practice (cGMP). One of ordinary skill in theart can identify other methods that can be used to prepare an enrichedpopulation of NK cells.

In some embodiments, enriched NK cells (typically >99% CD3 negativeand >85% CD56+) are expanded in vitro prior to or after geneticallyengineering the cells.

In one non-limiting example, the enriched NK cells are cultured with anirradiated EBV-LCL feeder cell line (SMI-LCL) in X-VIVO™ 20 medium(Lonza, Basel, Switzerland) with 10% human AB serum and 500 IU/ml ofinterleukin-2 (IL-2), for up to 21 days. Utilizing this technique,expansions of NK cells in the range of 200- to 1000-fold may be achieved(expanded NK cells are typically >99% CD3 negative and >90% CD56+). Insome examples, the starting population of enriched NK cells is about0.8-1.6×10⁸ total NK cells, which over a 2-4 week period expand up to1000-fold or greater in vitro. Similar numbers of NK cells have beenexpanded in scaled up experiments using GMP conditions. In someexamples, NK cells are expanded in G-Rex® containers (Wilson Wolf, NewBrighton, Minn.). The G-Rex®100 container support NK expansions to dosesof 2.5×10⁸ NK cells/kg or higher. NK cells cultured in G-Rex®100containers could be cultured at concentrations up to 4×10⁶ NK cells/ml.

In some embodiments, bulk NK cells or NK cells subsets isolated byadditional enriching procedures, such as through the use ofimmune-magnetic beads or flow sorting, may be grown in cell culturemedium, e.g., Cellgro SCGM serum-free media (CellGenix, Gaithersburg,Md.) containing 10% human AB serum, 50 U/mL penicillin, 50 gig/mLstreptomycin, and 500 IU/mL IL-2 or in X-VIVO™ 20 media containing 10%heat inactivated human AB serum or 10% autologous serum.

Non-expanded and expanded NK cells can be analyzed by flow cytometry forthe expression of markers such as CD56, CD16, TRAIL, FasL, NKG2D, LFA-1,perforin, and granzymes A and B. In some examples, expression of one ormore of the markers is measured at baseline and >10 days following invitro expansion. Chromium release assays can be used to assess fresh vs.expanded NK cell cytotoxicity against cancer cell targets. One ofordinary skill in the art can identify other methods to assess the NKcell population (for example, purity), viability, and/or activity.

In some embodiments, expand primary cells derived from a subject may beexpanded and/or cultured before genetic engineering. In some embodimentsthe engineered primary cells are cultured and/or expanded followingengineering and prior to administration to a patient.

NK Cell Line

In some embodiments, NK cell lines can be engineered as describedherein. In some embodiments, the engineering NK cells include engineeredNK cell lines. In some aspects, engineered cell lines allow productionof higher amounts of cells without having to expand small numbers of NKcells that are derived from a subject. Engineered cell lines also havethe advantage of being well characterized.

In some embodiments, the cell line is a clonal cell line. In someembodiments, the cell line is derived from a patient with NK-cellleukemia or lymphoma. In some embodiments, the NK cell line comprisesNK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1.

The NK-92 is a NK-like cell line that was initially isolated from theblood of a subject suffering from a large granular lymphoma andsubsequently propagated in cell culture. The NK-92 cell line has beendescribed (Gong et al., 1994; Klingemann, 2002). NK-92 cells have aCD3−/CD56+ phenotype that is characteristic of NK cells. They expressall of the known NK cell-activating receptors except CD16, but lack allof the known NK cell inhibitory receptors except NKG2A/CD94 andILT2/LIR1, which are expressed at low levels. Furthermore, NK-92 is aclonal cell line that, unlike the polyclonal NK cells isolated fromblood, expresses these receptors in a consistent manner with respect toboth type and cell surface concentration. Similarly, NK-92 cells are notimmunogenic and do not elicit an immune rejection response whenadministered therapeutically to a human subject. Indeed NK-92 cells arewell tolerated in humans with no known detrimental effects on normaltissues.

Some existing cell lines, such as NK-92 do not naturally express CD16.In some embodiments, the method provided herein includes engineering aNK cell line, such as NK-92, to express CD16. Klingemann et al., alsodiscuss the advantages of the NK92 cell lines. Klingemann et al,Frontiers in Immunology, vol 7, article 91 pp 1-7 (2016) In someembodiments, the methods provided herein include engineering a cell lineto express CD3ζ. In some embodiments CD16 and/or CD3ζ can be expressedinducibly or transiently. In such embodiments, the cell line is notengineered to heterologously or recombinantly express FcRγ chain or isengineered to reduce or eliminate expression of FcRγ chain in the cell.

Methods of Producing Engineered NK Cells

Provided herein are methods of producing an engineered NK cell that isgenetically engineered to reduce FcRγ chain expression, activity and/orsignaling in the cell as described. For example, the method may compriseintroducing a genetic disruption of a gene encoding FcRγ chain, a geneencoding a protein that regulates expression or activity of FcRγsignaling adaptor (e.g. a transcription factor, such as PLZF or HELIOS)and/or a gene encoding a protein that is involved in FcRγ-mediatedsignaling (e.g. a downstream signaling molecule, such as SYK, DAP2 orEAT2) as described. In some embodiments, the method may compriseintroducing an inhibitory nucleic acid molecule that targets a geneencoding FcRγ chain, a gene encoding a protein that regulates expressionor activity of FcRγ signaling adaptor (e.g. a transcription factor, suchas PLZF or HELIOS) and/or a gene encoding a protein that is involved inFcRγ-mediated signaling (e.g. a downstream signaling molecule, such asSYK, DAP2 or EAT2) as described.

In some embodiments, the method provided herein comprises isolating anNK cell from a subject, such as by the methods as described above orknown to a skilled artisan, and reducing the expression of FcRγ chainexpression, activity and/or signaling in the cell in accord with theprovided methods. In some embodiments, the method provided hereincomprises obtaining an NK cell line, such as any described herein, andengineering the cell to reduce expression of FcRγ expression, activityand/or signaling in the cell in accord with the provided methods.

One of ordinary skill in the art will appreciate that there are manyways of decreasing the expression or activity of FcRγ. For example, thelevel of transcription can be decreased. One method of decreasing geneexpression, such as FcRγ chain expression, involves modifying anendogenous gene to decrease transcription. For example, the FcRγ chaingene may be deleted, disrupted, or mutated. In addition to targeting theFcRγ RNA, mutating, or modifying the FcRγ gene, FcRγ protein level canbe decreased by effecting a molecule that increases FcRγ gene expressionor activity, such as a transcription factor that regulates transcriptionof FcRγ. In some embodiments a gene that regulates transcription ortranslation of the FcRγ chain gene may be deleted, disrupted, ormutated. In some of these embodiments, the gene is a transcriptionfactor that regulates expression of the FcRγ chain gene. Specifically,inhibition of a transcription factor that positively regulates FcRγexpression will result in decreased FcRγ expression. Transcriptionfactors that regulate FcRγ transcription include HELIOS and PLZF.

One of ordinary skill in the art will understand that there are manysuitable methods for disrupting FcRγ chain gene or other gene, such asthose described herein. For example, the entire gene locus, such as FcRγlocus, may be deleted. In some cases, it is also suitable to delete aportion of the gene, for example an exon, or a domain. Specifically, theITAM signaling domain of FcRγ may be deleted. Alternatively, theprovided methods also include introducing one or more amino acidsubstitutions into the gene locus, such as FcRγ locus, such as aninactivating mutation. In some embodiments, a stop codon can beintroduced into the mRNA, such as FcRγ mRNA, to produce a truncatedand/or inactivated form of the expressed gene, such as FcRγ signalingadaptor. In some embodiments, regulatory elements of the gene, such asFcRγ gene, can also be mutated or deleted in order to reduce expression,activity and/or signaling of FcRγ signaling adaptor.

In some embodiments, gene disruption can be carried out in mammaliancells using site-specific endonucleases. Endonucleases that allow forsite-specific deletion of a gene are well known in the art and mayinclude TAL nucleases, meganucleases, zinc-finger nucleases, Cas9, andArgonaute. Methods for producing engineered, site-specific endonucleasesare known in the art. The site-specific endonuclease can be engineeredto recognize and delete or modify a specific gene, such as the FcRγchain gene.

In one embodiment, zinc-finger nucleases (ZFNs) can be engineered torecognize and cut predetermined sites in a genome. ZFNs are chimericproteins comprising a zinc finger DNA-binding domain fused to thenuclease domain of the FokI restriction enzyme. The zinc finger domaincan be redesigned through rational or experimental means to produce aprotein which binds to a pre-determined DNA sequence, about orapproximately 18 basepairs in length. By fusing this engineered proteindomain to the FokI nuclease, it is possible to target DNA breaks withgenome-level specificity. ZFNs have been used extensively to target geneaddition, removal, and substitution in a wide range of eukaryoticorganisms (reviewed in S. Durai et al., Nucleic Acids Res 33, 5978(2005)).

In other embodiments, TAL-effector nucleases (TALENs) can be generatedto cleave specific sites in genomic DNA. Like a ZFN, a TALEN comprisesan engineered, site-specific DNA-binding domain fused to the FokInuclease domain (reviewed in Mak, et al. (2013) Curr Opin Struct Biol.23:93-9). In this case, however, the DNA binding domain comprises atandem array of TAL-effector domains, each of which specificallyrecognizes a single DNA basepair. Because ZFNs and TALENs areheterodimeric so that the production of a single functional nuclease ina cell requires co-expression of two protein monomers, compact TALENsprovide an alternative endonuclease architecture that avoids the needfor dimerization (Beurdeley, et al. (2013) Nat Commun. 4: 1762). ACompact TALEN comprises an engineered, site-specific TAL-effectorDNA-binding domain fused to the nuclease domain from the I-TevI homingendonuclease. Unlike FokI, I-TevI does not need to dimerize to produce adouble-strand DNA break so a Compact TALEN is functional as a monomer.

In some embodiments, engineered endonucleases based on the CRISPR/Cas9system are also known in the art and can be employed in the providedmethods to engineer the cells (Ran, et al. (2013) Nat Protoc.8:2281-2308; Mali et al. (2013) Nat Methods. 10:957-63). A CRISPRendonuclease comprises two components: (1) a caspase effector nuclease,typically microbial Cas9; and (2) a short “guide RNA” that directs thenuclease to a location of interest in the genome. In some embodiments,the guide RNA comprises an approximately 20 nucleotide targetingsequence. By expressing multiple guide RNAs in the same cell, eachhaving a different targeting sequence, it is possible to target DNAbreaks simultaneously to multiple sites in in the genome. Methods ofusing CRISPR-Cas9 are well known in the art.

In some aspects, the guide sequence is any polynucleotide sequencecomprising at least a sequence portion that has sufficientcomplementarity with a target polynucleotide sequence, such as a geneencoding FcRγ, PLZF, HELIOS, SYK, DAB2 or EAT2, to hybridize with thetarget sequence and direct sequence-specific binding of the CRISPRcomplex to the target sequence. Typically, in the context of formationof a CRISPR complex, “target sequence” generally refers to a sequence towhich a guide sequence is designed to have complementarity, wherehybridization between the target sequence and a guide sequence promotesthe formation of a CRISPR complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a CRISPR complex. In someembodiments, the degree of complementarity between a guide sequence andits corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some embodiments, a guidesequence is selected to reduce the degree of secondary structure withinthe guide sequence. Secondary structure may be determined by anysuitable polynucleotide folding algorithm.

In some embodiments, a CRISPR enzyme (e.g. Cas9 nuclease) in combinationwith (and optionally complexed with) a guide sequence is delivered tothe cell. In some embodiments, one or more elements of a CRISPR systemis derived from a type I, type II, or type III CRISPR system. In someembodiments, one or more elements of a CRISPR system are derived from aparticular organism comprising an endogenous CRISPR system, such asStreptococcus pyogenes or Staphylococcus aureus.

In the one embodiment of the invention, the DNA break-inducing agent isan engineered homing endonuclease (also called a “meganuclease”). Homingendonucleases are a group of naturally-occurring nucleases whichrecognize 15-40 base-pair cleavage sites commonly found in the genomesof plants and fungi. They are frequently associated with parasitic DNAelements, such as group 1 self-splicing introns and inteins. Theynaturally promote homologous recombination or gene insertion at specificlocations in the host genome by producing a double-stranded break in thechromosome, which recruits the cellular DNA-repair machinery (Stoddard(2006), Q. Rev. Biophys. 38: 49-95). Homing endonucleases are commonlygrouped into four families: the LAGLIDADG family, the GIY-YIG family,the His-Cys box family and the HNH family. These families arecharacterized by structural motifs, which affect catalytic activity andrecognition sequence. For instance, members of the LAGLIDADG family arecharacterized by having either one or two copies of the conservedLAGLIDADG motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18):3757-3774). The LAGLIDADG homing endonucleases with a single copy of theLAGLIDADG motif form homodimers, whereas members with two copies of theLAGLIDADG motif are found as monomers.

Another method of decreasing FcRγ chain expression, activity and/orsignaling involves introducing an inhibitory nucleic acid, such as aninhibitory RNA, into the cell that targets, e.g. is complementary to, atarget gene transcript, such as an FcRγ, PLZF, HELIOS, SYK, DAB2 or EAT2gene transcript, thereby reducing expression of the gene product. Forexample the nucleic acid may target FcRγ chain mRNA. In otherembodiments, the inhibitory nucleic acid may target the mRNA of a genethat regulates transcription or translation of the FcRγ chain gene, suchas a transcription factor, for example PLZF or HELIOS mRNA. In someembodiments the nucleic acid targets the mRNA of gene encoding a proteininvolved in FcRγ-mediated signaling, such as SYK, DAB2 or EAT-2 mRNA.

The presently disclosed subject matter takes advantage of RNAitechnology (for example shRNA, siRNA and miRNA molecules and ribozymes)to cause the down regulation of cellular genes, a process referred to asRNA interference (RNAi). As used herein, “RNA interference” (RNAi)refers to a process of sequence-specific post-transcriptional genesilencing mediated by a small interfering RNA (siRNA) or short hairpinRNA (shRNA) molecules, miRNA molecules or synthetic hammerheadribozymes. See generally Fire et al., Nature 391:806-811, 1998, and U.S.Pat. No. 6,506,559. The process of RNA interference (RNAi) mediatedpost-transcriptional gene silencing is thought to be an evolutionarilyconserved cellular defense mechanism that has evolved to prevent theexpression of foreign genes (Fire, Trends Genet 15:358-363, 1999).

In some embodiments, a recombinant virus comprising nucleic acidencoding the RNA can be produced. Engineering retroviral vectors isknown to those having ordinary skill in the art. Such a skilled artisanwould readily appreciate the multiple factors involved in selecting theappropriate virus and vector components needed to optimize recombinantvirus production for use with the presently disclosed subject matterwithout the necessity of further detailed discussion herein. As onenon-limiting example, a retrovirus can be engineered comprising DNAencoding an shRNA comprising an siRNA.

The gene expression may be reduced permanently, transiently, orinducibly. Suitable inducible systems are well known and includeeukaryotic promoters responsive to heavy metals, Lac/VP16, and thetetracycline repressor system.

On the other hand, it may be beneficial to permanently reduce expressionof the gene, for example by producing a cell line with a deletion,substitution, or insertion that causes inactivation of the gene.

Retroviral systems can be used to introduce cDNAs into NK cells. Methodsof eukaryotic cell transfection and prokaryotic cell transformation arewell known in the art. The choice of host cell dictates the preferredtechnique for introducing the polynucleotide of interest. Introductionof polynucleotides into an organism may also be done with ex vivotechniques that use an in vitro method of transfection, as well asestablished genetic techniques, if any, for that particular organism.

Other vectors and packaging cell lines have been used in the preparationof genetically modified variants of NK cells and can be usedequivalently herein. Retroviral transduction systems have also beensuccessfully used to transduce a variety of genes into NK cells. By wayof example, these alternative methods include, but are not limited tothe p-JET vector in conjunction with FLYA13 packaging cells (Gerstmayeret al., 1999), the plasmid-based kat retroviral transduction system, andDFG-hIL-2-neo/CRIP (Nagashima et al., 1998). Electroporation and “genegun” introduction of the vector into the packaging cells is alsopracticed. Use of the pBMN-IRES-EGFP vector in combination with thePhoenix-Amphotropic packaging cell line is convenient for the purpose ofthis and the following Examples in that it provides high efficiencies ofPhoenix-Amphotropic cell transfection; the use of Moloney LTR promotersresults in a high level of CD16 expression; the virus is produced athigh titers; the efficiency of NK transduction is improved over othervectors that have been used to transduce NK cells; and the vectorprovides adequate space to accommodate the CD16 cDNA or alternativeinserts. The pBMN-IRES-EGFP vector further incorporates genes forenhanced green fluorescent protein (EGFP), which can be used as anendogenous surrogate marker for gene expression. The Phoenix cell linestably expresses this vector in episomal form along with producing otherviral components, thus allowing the cells to stably produce virus for anextended period of time.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20 deg. C. Chloroform is used as the onlysolvent since it is more readily evaporated than methanol. “Liposome” isa generic term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise engineer the cell in accord with the providedmethods, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR or “biochemical” assays, such as detecting the presenceor absence of a particular peptide, e.g., by immunological means (ELISAsand Western blots).

Features of Engineered Cells

The expression of gene products associated with the engineering of genesas described can be assessed following or in connection with theengineering and/or culturing of the NK cells. Any available procedureknown to a skilled artisan can be employed to detect the gene productsso long as the procedure does not injure the cells. For example, the NKcells can be detected, identified and/or isolated by flow cytometry fordetection of a cell surface marker that correlates with expression ofthe gene product, such as that correlates with FcRγ expression. Thetargets for modulating expression by genetic engineering as described,such as FcRγ proteins, are intracellular proteins that are not easilydetected unless the cells are treated to allow intracellular proteins tobe detected, for example, by fixation and permeabilization.

In some embodiments, the methods provided herein result in expression ofa particular gene product in the engineered cell that is reduced bygreater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% as compared to the expression of the gene productin the NK cell that is not genetically engineered. In some embodiments,the methods provided herein reduce FcRγ chain expression by greater than10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%as compared to the expression of FcRγ chain in the NK cell that is notgenetically engineered. In some of these embodiments, the level of FcRγchain expression is reduced to an undetectable level using an immunoblotassay.

Expression of gene products as described, such as FcRγ chain expression,can be measured in a variety of ways. For example, RNA expression can bemeasured by northern blot, qPCR, and FISH. Protein expression can alsobe measured, for example using flow cytometry, western blot,immunohistochemistry, ELISA, and the like.

In some aspects, the provided engineered NK cells exhibit enhancedactivity when activated by antibody, such as can occur byantibody-mediated crosslinking of CD16 or by antibody-coated cells, e.g.antibody-coated tumor cells. In some embodiments, the providedengineered NK cells are particular responsive in the presence of anantibody or other Fc-containing protein and can be used in methods incombination with an administered monoclonal antibody or otherFc-containing protein specific to a tumor, virus or microbial cell. Insome cases, the provided engineered NK cells exhibit properties orfeatures that are the same as or similar to g-NK cells, which are aspecific subset of NK cells deficient in FcRγ present in small numbersin individuals, but only in about one-third of the population (see e.g.published patent appl. No. US2013/0295044; see also Hwang et al. (2012)Int. Immunol., 24:793-802 and Lee et al. (2015) Cell Immunity,42:431-442)).

In some embodiments, the increased activity is observed following CD16engagement by CD16 crosslinking, such as can occur in the presence of anantibody by binding of the Fc portion of the antibody to CD16 andinitiation of ADCC. In some embodiments, the increased activity can bedetermined by monitoring phosphorylation of the CD3ζ chain, signalingmolecules, CA2+ flux, expression or secretion of cytokines by the cell(e.g. IFN-gamma or TNF-α), expression or secretion of chemokines by theengineered cell (MIP-1α, MIP-1β or RANTES), degranulation response,expression of Granzyme B and/or cytotoxic killing response. Any of anumber of well-known assays can be used to assess the properties oractivities of the engineered NK cells (see e.g. Hwang et al. (2012) Int.Immunology, 24:793-802; published patent appl. No. US2013/0295044). Insome embodiments, the activity of the engineered NK cells following CD16crosslinking or engagement is increased by greater than or greater thanabout 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or more compared to in the activity inthe engineered NK cell in the same assay but in the absence of CD16crosslinking or engagement.

In vitro assays are commonly employed for assessing antibody-dependentcellular cytotoxicity (ADCC). In one example, target cells (e.g. cellsthat express an antigen that is appropriate to the antibody beingevaluated) are loaded with an indicator material (such as 51Cr), and theindicator-loaded target cells are treated with the antibody to beevaluated. The resulting cells are exposed to NK effector cells asdescribed herein. Lysis of the target cells is indicated by the releaseof the indicator material into the assay supernatant where itsconcentration can be measured by a suitable method such as scintillationcounting (51Cr) or fluorescence intensity or lifetime determination.Efficacy can likewise be assessed by the measurement of surrogateindicators such as cytokine release by the NK cells; the up-regulationof NK cell activation markers, such as CD25, CD69 and/or CD95L;activation of NK cell transcription factors, such as NF-AT or NF-κB; orthe activation of caspases or other markers of apoptosis in the targetcells. Parental NK cells (such as non-genetically engineered NK cells)serve as a control because they permit differentiating betweenADCC-mediated cytotoxicity and other cytolytic effects that NK cellsexert on the target cells.

In some embodiments, the engineered NK cells are able to persist in anindividual for an extended period and therefore the number of times thatthe cells need to be administered to have a therapeutic effect can bedecreased. In some embodiments, the engineered cells provided hereinpersist for at least one month, at least two months, at least threemonths, at least four months, at least five months, at least six months,or at least months after administration.

III. Compositions and Kits Comprising Engineered NK Cells

Provided herein are compositions comprising the provided engineered NKcells. Among the compositions are pharmaceutical compositions andformulations for administration, such as for adoptive cell therapy. Insome embodiments, the engineered cells are formulated with apharmaceutically acceptable carrier.

A pharmaceutically acceptable carrier can include all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration (Gennaro, 2000, Remington: The science andpractice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia,Pa.). Examples of such carriers or diluents include, but are not limitedto, water, saline, Ringer's solutions, dextrose solution, and 5% humanserum albumin. Liposomes and non-aqueous vehicles such as fixed oils mayalso be used. Supplementary active compounds can also be incorporatedinto the compositions. The pharmaceutical carrier should be one that issuitable for NK cells, such as a saline solution, a dextrose solution ora solution comprising human serum albumin.

In certain embodiments, the number of cells in the composition providesthe engineered NK cells at a therapeutically effective amount. In someembodiments, the amount is an amount that reduces the severity, theduration and/or the symptoms associated with cancer, viral infection,microbial infection, or septic shock in an animal. In certain otherembodiments, a therapeutically effective amount is a dose of cells thatresults in a reduction of the growth or spread of cancer by at least2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least35%, at least 45%, at least 50%, at least 75%, at least 85%, by at least90%, at least 95%, or at least 99% in a patient or an animaladministered a composition described herein relative to the growth orspread of cancer in a patient (or an animal) or a group of patients (oranimals) not administered the composition. In some embodiments, aneffective amount for cytotoxicity is defined as amount of engineered NKcells that is able to inhibit or reduce the growth of cancer, viral andmicrobial cells. In some embodiments, the composition comprises a doseof engineered NK cells that is from or from about 10⁵ to about 10¹²cells, or about 10⁵ to about 10⁸ cells, or about 10⁶ to about 10¹²cells, or about 10⁸ to about 10¹ cells, or about 10⁹ to about 10¹⁰cells. In some embodiments, the composition comprises greater than orgreater than about 10⁵, about 10⁶, about 10⁷, about 10⁸, about 10⁹,about 10¹⁰, about 10¹¹, or about 10¹² cells.

In some embodiments, the volume of the composition is at least or atleast about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL or 500 mL, suchas is from or from about 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL,100 mL to 500 mL, 100 mL to 200 mL or 200 mL to 500 mL, each inclusive.In some embodiments, the composition has a cell density of at least orat least about 1×10⁵ cells/mL, 5×10⁵ cells/mL, 1×10⁶ cells/mL, 5×10⁶cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL or 1×10⁸ cells/mL. In someembodiment, the cell density of the composition is between or betweenabout 1×10⁵ cells/mL to 1×10⁸ cells/mL, 1×10⁵ cells/mL to 1×10⁷cells/mL, 1×10⁵ cells/mL to 1×10⁶ cells/mL, 1×10⁶ cells/mL to 1×10⁷cells/mL, 1×10⁶ cells/mL to 1×10⁸ cells/mL, 1×10⁶ cells/mL to 1×10⁷cells/mL or 1×10⁷ cells/mL to 1×10⁸ cells/mL, each inclusive.

Depending upon the method of engineering the NK cell, it may benecessary or desirable to culture the NK cells to expand them prior toformulating them as a composition for administration. In some of theembodiments, methods of producing a composition comprising engineered NKcells comprises culturing or incubating the engineered NK cells, such asto expand the cells to a therapeutically effective amount prior toadministering the NK cells to an individual in need thereof.

Suitable methods for culturing and expanding NK cells are known. Forexample, the NK cells may be cultured using feeder cells, or in thepresence of cytokines to enhance their growth and/or activation. As usedherein “culturing” includes providing the chemical and physicalconditions (e.g., temperature, gas) which are required for NK cellmaintenance, and growth factors. In one embodiment, culturing the NKcells includes providing the NK cells with conditions for proliferation.Examples of chemical conditions which may support NK cell proliferationinclude but are not limited to buffers, nutrients, serum, vitamins andantibiotics as well as cytokines and other growth factors which aretypically provided in the growth (i.e., culture) medium. In oneembodiment, the NK culture medium includes MEMa comprising 10% FCS orCellGro SCGM (Cell Genix) comprising 5% Human Serum/LiforCell® FBSReplacement (Lifeblood Products). Other media suitable for use with theinvention include, but are not limited to Glascow's medium (GibcoCarlsbad Calif.), RPMI medium (Sigma-Aldrich, St Louis Mo.) or DMEM(Sigma-Aldrich, St Louis Mo.). It will be noted that many of the culturemedia contain nicotinamide as a vitamin supplement for example, MEMα(8.19 μM nicotinamide), RPMI (8.19 μM nicotinamide), DMEM (32.78 μMnicotinamide) and Glascow's medium (16.39 μM nicotinamide).

In some embodiments, such as for applications in which cells areintroduced (or reintroduced) into a human subject, culturing is carriedout using serum-free formulations, such as AIM V™ serum free medium forlymphocyte culture or MARROWMAX™ bone marrow medium. Such mediumformulations and supplements are available from commercial sources suchas Invitrogen (GIBCO) (Carlsbad, Calif.). The cultures can besupplemented with amino acids, antibiotics, and/or with cytokines topromote optimal viability, proliferation, functionality and/or andsurvival.

In some embodiments, culturing the population of cells comprising theengineered NK cells is effected without a feeder layer or feeder cells.In some of these embodiments, the engineered NK cells can be culturedwith a growth factor. According to some embodiments, the at least onegrowth factor comprises a growth factor selected from the groupconsisting of SCF, FLT3, IL-2, IL-7, IL-15, IL-12 and IL-21. Accordingto some embodiments, the at least one growth factor is IL-2 or IL-2 andIL-15. According to some embodiments, the at least one growth factor issolely IL-2.

In some embodiments, the provided compositions include those in whichthe genetically engineered NK cells, such as engineered NK cells thatare reduced for FcRγ chain expression, activity and/or signaling in thecell, make up at least or at least about 60%, 70%, 80%, 85%, 90%, 95% ormore of the cells in the composition or of the NK cells in thecomposition.

Also provided herein are compositions that are suitable forcryopreserving engineered NK cells. In some embodiments, the compositioncomprises an engineered NK cell and a cryoprotectant. In someembodiments, the cryoprotectant is or comprises DMSO and/or s glycerol.In some embodiments, compositions formulated for cryopreservation can bestored at low temperatures, such as ultra low temperatures, for example,storage with temperature ranges from −40° C. to −150° C., such as orabout 80° C.±6.0° C.

In some embodiments, the engineered NK cells can be preserved at ultralow temperature before the administration to a patient. The engineeredNK cells can also be preserved at ultra low temperature after isolationfrom a mammalian subject and prior to the genetic engineering. Forexample, lymphocytes or another source of engineered NK cells can beisolated, stored at ultra low temperature and then processed to yieldengineered NK cells. Alternatively, the lymphocytes or another source ofengineered NK cells can be isolated, processed to yield engineered NKcells and then stored at ultra-low temperature.

A typical method for the preservation at ultra low temperature in smallscale is described, for example, in U.S. Pat. No. 6,0168,991. Forsmall-scale, cells can be preserved at ultra low temperature by lowdensity suspension (e.g., at a concentration of about 200×106/ml) in 5%human albumin serum (HAS) which is previously cooled. An equivalentamount of 20% DMSO can be added into the HAS solution. Aliquots of themixture can be placed into vials and frozen overnight inside an ultralow temperature chamber at about −80° C.

In some embodiments, the cryopreserved NK cells are prepared foradministration by thawing. In some cases, the NK cells can beadministered to a subject immediately after thawing. In such anembodiment, the composition is ready-to-use without any furtherprocessing. In other cases, the NK cells are further processed afterthawing, such as by resuspension with a pharmaceutically acceptablecarrier, incubation with an activating or stimulating agent, or areactivated washed and resuspended in a pharmaceutically acceptable bufferprior to administration to a subject.

Kits comprising engineered cells are also provided herein. For example,in some embodiment provided herein is a kit comprising an engineeredcell and an additional agent. In some embodiments, the additional agentcomprises an Fc domain. In some embodiment the additional agent is an Fcfusion protein or an antibody. In some embodiments, the additional agentis a human, humanized, or chimeric antibody. In some of theseembodiments, the additional agent is a full length antibody. Exemplaryantibodies are described below.

IV. Methods of Treatment

In some embodiments, provided herein is a method of treating a conditionin an individual, comprising administering engineered NK cells to anindividual in need thereof.

In some embodiments, the method comprises administering an effectiveamount of engineered cells to an individual. In some embodiments, fromor from about 10⁵ to about 10¹² cells, or about 10⁵ to about 10⁸ cells,or about 10⁶ to about 10¹² cells, or about 10⁸ to about 10¹¹ cells, orabout 10⁹ to about 10¹⁰ cells. In some embodiments, the compositioncomprises, about 10⁵, about 10⁶, about 10⁷, about 108, about 10⁹, about10¹⁰, about 10¹¹, or about 10¹² cells are administered to theindividual. In some embodiments, from or from about 10⁶ to 10¹⁰engineered NK cells/kg are administered to the subject.

In some embodiments, the engineered NK cells are administered to anindividual soon after isolation and the engineering of the NK cells. Insome embodiments, the engineered NK cells are administered to anindividual within 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days of isolationand the engineering.

In other embodiments, the engineered NK cells are stored or expanded bygrowth in culture prior to administration and/or engineering, such as bymethods described above. For example, the NK cells can be stored forgreater than 6, 12, 18, or 24 months prior to engineering and/oradministration to the individual.

In some cases, clonal cell lines of NK cells are derived from cancerouscells and thus may divide out of control upon administration to apatient. In some embodiments, the engineered NK cells, such as thosefrom clonal cell lines, may be irradiated prior to administration to asubject to prevent them from dividing out of control.

The engineered NK cells can be can be administered to a subject by anyconvenient route including parenteral routes such as subcutaneous,intramuscular, intravenous, and/or epidural routes of administration.

The provided engineered NK cells and compositions can be used in methodsof treating an individual with a tumor or hyperproliferative disordersor microbial infection such as a viral infection, yeast infection,fungal infection, protozoan infection and/or bacterial infection. Thedisclosed methods of treating a subject with the engineered cells can bein combination with a therapeutic monoclonal antibody, such as ananti-tumor antigen or anti-cancer antibody, anti-viral antibody oranti-bacterial antibody. The engineered NK cells can be administered fortreatment of animals, such as mammalian animals, for example humansubjects.

In some examples, the methods include treating a hyperproliferativedisorder, such as a hematological malignancy or a solid tumor. Examplesof types of cancer and proliferative disorders that can be treated withthe compositions described herein include, but are not limited to,leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia, chronic myelocytic (granulocytic) leukemia, and chroniclymphocytic leukemia), lymphoma (e.g., Hodgkin's disease andnon-Hodgkin's disease), fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma,Ewing's tumor, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, renal cell carcinoma, hepatoma, Wilm's tumor,cervical cancer, uterine cancer, testicular tumor, lung carcinoma, smallcell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma,dysplasia and hyperplasia. The treatment and/or prevention of cancerincludes, but is not limited to, alleviating one or more symptomsassociated with cancer, the inhibition or reduction of the progressionof cancer, the promotion of the regression of cancer, and/or thepromotion of the immune response.

In some examples, the methods include treating a viral infection, suchas an infection caused by the presence of a virus in the body. Viralinfections include chronic or persistent viral infections, which areviral infections that are able to infect a host and reproduce within thecells of a host over a prolonged period of time-usually weeks, months oryears, before proving fatal. Viruses giving rise to chronic infectionsthat which may be treated in accordance with the present inventioninclude, for example, the human papilloma viruses (HPV), Herpes simplex,and other herpes viruses, the viruses of hepatitis B and C as well asother hepatitis viruses, human immunodeficiency virus, and the measlesvirus, all of which can produce important clinical diseases. Prolongedinfection may ultimately lead to the induction of disease which may be,e.g., in the case of hepatitis C virus liver cancer, fatal to thepatient. Other chronic viral infections which may be treated inaccordance with the present invention include Epstein Barr virus (EBV),as well as other viruses such as those which may be associated withtumors.

Examples of viral infections which can be treated or prevented with thecompositions and methods described herein include, but are limited to,viral infections caused by retroviruses (e.g., human T-celllymphotrophic virus (HTLV) types I and II and human immunodeficiencyvirus (HIV)), herpes viruses (e.g., herpes simplex virus (HSV) types Iand II, Epstein-Ban virus and cytomegalovirus), arenaviruses (e.g.,lassa fever virus), paramyxoviruses (e.g., morbillivirus virus, humanrespiratory syncytial virus, and pneumovirus), adenoviruses,bunyaviruses (e.g., hantavirus), cornaviruses, filoviruses (e.g., Ebolavirus), flaviviruses (e.g., hepatitis C virus (HCV), yellow fever virus,and Japanese encephalitis virus), hepadnaviruses (e.g., hepatitis Bviruses (HBV)), orthomyoviruses (e.g., Sendai virus and influenzaviruses A, B and C), papovaviruses (e.g., papillomaviruses),picornaviruses (e.g., rhinoviruses, enteroviruses and hepatitis Aviruses), poxviruses, reoviruses (e.g., rotaviruses), togaviruses (e.g.,rubella virus), and rhabdoviruses (e.g., rabies virus). The treatmentand/or prevention of a viral infection includes, but is not limited to,alleviating one or more symptoms associated with said infection, theinhibition, reduction or suppression of viral replication, and/or theenhancement of the immune response.

In some embodiments, the compositions are used in a method of treating ayeast or bacterial infection. For example, the compositions and methodsdescribed herein can treat infections relating to Streptococcuspyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseriameningitidis, Corynebacterium diphtheriae, Clostridium botulinum,Clostridium perfringens, Clostridium tetani, Haemophilus influenzae,Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis,Staphylococcus aureus, Vibrio cholera, Escherichia coli, Pseudomonasaeruginosa, Campylobacter (Vibrio) fetus, Campylobacterjejuni, Aeromonashydrophila, Bacillus cereus, Edwardsiella tarda, Yersiniaenterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigelladysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium,Treponema pallidum, Treponemapertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii,Francisella tularensis, Brucella aborts, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsiaprowazeki, Rickettsiatsutsugumushi, Chlamydia spp., Helicobacter pylori or combinationsthereof.

V. Combination Therapy

In some embodiments, the presently engineered NK cells exhibit enhancedactivity when activated by antibodies or Fc-containing proteins. Forexample, the engineered cells can be activated by antibody-mediatedcrosslinking of CD16 or by antibody-coated tumor cells.

In some embodiments, provided herein is a method of treating a conditionin an individual comprising administering an engineered NK cell and anantibody. One of ordinary skill in the art can select an appropriatetherapeutic (e.g., anti-cancer) monoclonal antibody to administer to thesubject with the engineered NK cells described herein, such as dependingon the particular disease or condition of the individual. Suitableantibodies may include polyclonal, monoclonal, fragments (such as Fabfragments), single chain antibodies and other forms of specific bindingmolecules.

Abs may further comprise humanized or human Abs. Humanized forms ofnon-human Abs are chimeric Igs, Ig chains or fragments (such as Fv, Fab,Fab′, F(ab′)2 or other antigen-binding subsequences of Abs) that containminimal sequence derived from non-human Ig. In some embodiments, theantibody comprises an Fc domain.

Generally, a humanized antibody has one or more amino acid residuesintroduced from a non-human source. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization is accomplished bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody (Jones et al., 1986; Riechmann et al.,1988; Verhoeyen et al., 1988). Such “humanized” Abs are chimeric Abs(1989), wherein substantially less than an intact human variable domainhas been substituted by the corresponding sequence from a non-humanspecies. In practice, humanized Abs are typically human Abs in whichsome CDR residues and possibly some Fc residues are substituted byresidues from analogous sites in rodent Abs. Humanized Abs include humanIgs (recipient antibody) in which residues from a complementarydetermining region (CDR) of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit, having the desired specificity, affinity and capacity. In someinstances, corresponding non-human residues replace Fv frameworkresidues of the human Ig. Humanized Abs may comprise residues that arefound neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanized antibody comprisessubstantially all of at least one, and typically two, variable domains,in which most if not all of the CDR regions correspond to those of anon-human Ig and most if not all of the FR regions are those of a humanIg consensus sequence. The humanized antibody optimally also comprisesat least a portion of an Ig constant region (Fc), typically that of ahuman Ig (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

Human Abs can also be produced using various techniques, including phagedisplay libraries (Hoogenboom et al., 1991; Marks et al., 1991) and thepreparation of human mAbs (Boerner et al., 1991; Reisfeld and Sell,1985). Similarly, introducing human Ig genes into transgenic animals inwhich the endogenous Ig genes have been partially or completelyinactivated can be exploited to synthesize human Abs. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire (1997a; 1997b; 1997c; 1997d; 1997; 1997; Fishwild etal., 1996; 1997; 1997; 2001; 1996; 1997; 1997; 1997; Lonberg and Huszar,1995; Lonberg et al., 1994; Marks et al., 1992; 1997; 1997; 1997).

Specifically, the cells of the present invention can be targeted totumors by administration with an antibody that recognizes a tumorassociated antigen. One of ordinary skill in the art will appreciatethat the present engineered NK cells are suitable for use with a widevariety of antibodies that recognize tumor associated antigens.Non-limiting examples of a tumor associated antigen includes CD19, CD20,CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140,EpCAM, CEA, gpA33, mesothelin, α-fetoprotein, Mucin, PDGFR-alpha,TAG-72, CAIX, PSMA, folate-binding protein, scatter factor receptorkinase, a ganglioside, cytokerain, frizzled receptor, VEGF, VEGFR,Integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3,fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-alpha,phosphatidylserine, Syndecan 1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL,FAP, vimentin or tenascin. In some cases, the antibody is an anti-CD20antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-EGFRantibody and an anti-CD38 antibody. Exemplary antibodies includerituximab, trastuzumab, aletuzumab, certuximab, daratumumab, veltuzumab,ofatumumab, ublituximab, ocaratuzumab. Antibodies specific for aselected cancer type can be chosen, and include any antibody approvedfor treatment of cancer. Examples include trastuzumab (Herceptin) forbreast cancer, rituximab (Rituxan) for lymphoma, and cetuximab (Erbitux)for head and neck squamous cell carcinoma. A skilled artisan is familiarwith FDA-approved monoclonal antibodies able to bind particular tumor ordisease antigens, any of which can be used in accord with the providedmethods for treating the tumor or disease.

The engineered NK cells and the additional agent can be administeredsequentially or simultaneously. In some embodiments, the additionalagent can be administered before administration of the engineered NKcells. In some embodiments, the additional agent can be administeredafter administration of the NK cells. For example, the engineered NKcells can be administered simultaneously with antibodies specific for aselected cancer type. Alternatively, the engineered NK cells can beadministered at selected times that are distinct from the times whenantibodies specific for a selected cancer type are administered.

In particular examples, the subject is administered an effective dose ofan antibody before, after, or substantially simultaneously with thepopulation of engineered NK cells. In some examples, the subject isadministered about 0.1 mg/kg to about 100 mg/kg of the antibody (such asabout 0.5-10 mg/kg, about 1-20 mg/kg, about 10-50 mg/kg, about 20-100mg/kg, for example, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 8 mg/kg, about 10 mg/kg,about 16 mg/kg, about 20 mg/kg, about 24 mg/kg, about 36 mg/kg, about 48mg/kg, about 60 mg/kg, about 75 mg/kg, or about 100 mg/kg). An effectiveamount of the antibody can be selected by a skilled clinician, takinginto consideration the particular antibody, the particular disease orconditions (e.g. tumor or other disorder), the general condition of thesubject, any additional treatments the subject is receiving or haspreviously received, and other relevant factors. The subject is alsoadministered a population of modified NK cells described herein. Boththe antibody and the population of modified NK cells are typicallyadministered parenterally, for example intravenously; however, injectionor infusion to a tumor or close to a tumor (local administration) oradministration to the peritoneal cavity can also be used. One of skillin the art can determine appropriate routes of administration.

The engineered NK cells can also be administered simultaneously orsequentially with anti-microbial, anti-viral and other therapeuticagents. In some embodiments of the present invention, the engineeredcells can be administered to an individual in combination with cytokinesand/or growth factors. According to some embodiments of the presentinvention the at least one growth factor comprises a growth factorselected from the group consisting of SCF, FLT3, IL-2, IL-7, IL-15,IL-12 and IL-21. In some embodiments, the engineered NK cells and thecytokines or growth factors are administered sequentially. For example,the engineered NK cells may be administered first, followed byadministration of the cytokines and/or growth factors. In someembodiments, the engineered NK cells are administered simultaneouslywith the cytokines or growth factors.

In some embodiments, the subject is administered one or more cytokines(such as IL-2, IL-15, IL-21, and/or IL-12) to support survival and/orgrowth of NK cells. The cytokine(s) can be administered before, after,or substantially simultaneously with the NK cells. In some examples, thecytokine(s) can be administered after the NK cells. In one specificexample, the cytokine(s) is administered to the subject within about 1-8hours (such as within about 1-4 hours, about 2-6 hours, about 4-6 hours,or about 5-8 hours) of the administration of the NK cells.

In some embodiments, the provided methods also can include administeringengineered NK cells to an individual in combination with achemotherapeutic agent. In some embodiments, the chemotherapeutic agentmay comprise cyclophosphamide, fludarabine, methyl prednasone In someembodiments, the chemotherapeutic agent is selected from the groupconsisting of: thalidomide, cisplatin (cis-DDP), oxaliplatin,carboplatin, anthracenediones, mitoxantrone; hydroxyurea,methylhydrazine derivatives, procarbazine (N-methylhydrazine, MM),adrenocortical suppressants, mitotane (.omicron., .rho.′-DDD),aminoglutethimide, RXR agonists, bexarotene, tyrosine kinase inhibitors,imatinib, mechlorethamine, cyclophosphamide, ifosfamide, melphalan(L-sarcolysin), chlorambucil, ethylenimines, methylmelamines,hexamethylmelamine, thiotepa, busulfan, carmustine (BCNU), semustine(methyl-CCNTJ), lomustine (CCNU), streptozocin (streptozotocin), DNAsynthesis antagonists, estramustine phosphate, triazines, dacarbazine(OTIC, dimethyl-triazenoimidazolecarboxamide), temozolomide, folic acidanalogs, methotrexate (amethopterin), pyrimidine analogs, fiuorouracin(5-fluorouracil, 5-FU, 5FTJ), floxuridine (fluorodeox>′uridine, FUdR),cytarabine (cytosine arabinoside), gemcitabine, purine analogs,mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine,TG), pentostatin (2′-deoxycoformycin, deoxycoformycin), cladribine andfludarabine, topoisomerase inhibitors, amsacrine, vinca alkaloids,vinblastine (VLB), vincristine, taxanes, paclitaxel, protein boundpaclitaxel (Abraxane®), docetaxel (Taxotere®); epipodophyllotoxins,etoposide, teniposide, camptothecins, topotecan, irinotecan,dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin),doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin,buserelin, adrenocorticosteroids, prednisone, progestins,hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, diethylstilbestrol, ethinyl estradiol, tamoxifen, anastrozole;testosterone propionate, fluoxymesterone, flutamide, bicalutamide, andleuprolide.

In some embodiments, the cancer drug is thalidomide or its derivatives.In some embodiments, the cancer drug is selected from the groupconsisting of cisplatin, carboplatin, and oxaliplatin. In certainembodiments, the cancer drug is selected from the group consisting ofpaclitaxel, Abraxane®, and Taxotere®. In one embodiment, thechemotherueptic agent is selected from the group consisting ofasparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin,etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and6-mercaptopurine, cyclophosphamide, paclitaxel, and gemcitabine.

VI. Exemplary Embodiments

Among the provided embodiments herein are:

1. An engineered natural killer (NK) cell, wherein the NK cell isgenetically engineered to reduce FcRγ chain expression, activity and/orsignaling in the cell.

2. The engineered NK cell of embodiment 1, wherein the engineered NKcell comprises:

a genetic disruption of a gene encoding FcRγ chain and/or a geneticdisruption resulting in reduced expression of FcRγ chain in theengineered NK cell;

a genetic disruption of a gene encoding a protein regulating expressionor activity of FcRγ chain and/or a genetic disruption resulting inreduced expression of a protein regulating expression or activity ofFcRγ chain; and/or

a genetic disruption of a gene encoding a protein involved in FcRγchain-dependent signaling and/or a genetic disruption resulting inreduced expression of a protein involved in FcRγ chain-dependentsignaling.

3. The engineered NK cell of embodiment 2, wherein the geneticdisruption comprises a deletion, mutation and/or insertion resulting ina premature stop codon in the gene or a frameshift of the open readingframe of the gene.

4. The engineered NK cell of embodiment 2 or embodiment 3, wherein bothalleles of the gene encoding FcRγ chain, the gene encoding a proteinregulating expression or activity of FcRγ chain and/or the gene encodinga protein involved in FcRγ chain-dependent signaling are disrupted inthe engineered NK cells.

5. The engineered NK cell of embodiment 1, wherein the engineered NKcell comprises an inhibitory nucleic acid molecule targeting a gene inthe NK cell resulting in reduced expression of FcRγ chain, reducedexpression of a protein regulating expression or activity of FcRγ chainand/or reduced expression of a protein involved in FcRγ chain-dependentsignaling.

6. The engineered NK cell of any of embodiments 2-5, wherein theexpression of FcRγ chain, a protein regulating expression or activity ofFcRγ chain and/or a protein involved in FcRγ chain-dependent signalingis reduced by greater than or greater than about 50%, 60%, 70%, 80%,90%, or 95% as compared to the expression of the protein in the NK cellthat is not genetically engineered.

7. The engineered NK cell of any of embodiments 2-6, wherein expressionof a protein regulating expression or activity of FcRγ chain is reducedin the engineered NK cell and the protein is a transcription factor.

8. The engineered NK cell of embodiment 7, wherein the transcriptionfactor is PLZF (ZBTB16) or HELIOS (IKZF2).

9. The engineered NK cell of any of embodiments 2-6, wherein theexpression of a protein involved in FcRγ chain-dependent signaling isreduced in the engineered NK cell and the protein is a downstreamsignaling molecule.

10. The engineered NK cells of embodiment 9, wherein the downstreamsignaling molecule is SYK, DAB2 or EAT-2.

11. The engineered NK cell of any of embodiments 2-6, wherein expressionof FcRγ chain is reduced in the engineered cell.

12. An engineered NK cell comprising a genetic disruption in a geneencoding FcRγ chain, wherein expression of FcRγ is reduced in the cell.

13. The engineered NK cell of embodiment 12, wherein the geneticdisruption comprises a deletion, mutation and/or insertion resulting ina premature stop codon in the gene or a frameshift of the open readingframe of the gene.

14. The engineered NK cell of embodiment 12 or embodiment 13, whereinboth alleles of the gene encoding FcRγ chain are disrupted in the genomeof the engineered NK cell.

15. The engineered NK cell of any of embodiments 1, 5 or 6, wherein theengineered NK cell comprises an inhibitory nucleic acid molecule thattargets a gene encoding FcRγ chain.

16. The engineered NK cell of embodiment 15, wherein the inhibitorynucleic acid molecule comprises a sequence complementary to the geneencoding FcRγ chain.

17. The engineered NK cell of any of embodiments 11-16, wherein theexpression of FcRγ chain is reduced by greater than or greater thanabout 50%, 60%, 70%, 80%, 90%, or 95% as compared to the expression inthe NK cell that is not genetically engineered.

18. The engineered NK cell of any of embodiments 5-6 and 15-17, whereinthe inhibitory nucleic acid comprises an RNA interfering agent.

19. The engineered NK cell of any of embodiments 5-6 and 15-18, whereinthe inhibitory nucleic acid comprises siRNA, shRNA, or miRNA.

20. The engineered NK cell of any of embodiments 1-19, wherein thereduced expression, activity and/or signaling of FcRγ is permanent,transient or inducible.

21. The engineered NK cell of any of embodiments 1-20, wherein theexpression, activity and/or signaling of FcRγ chain is reduced bygreater than or greater than about 50%, 60%, 70%, 80%, 90%, or 95% ascompared to the expression, activity and/or signaling in the NK cellthat is not genetically engineered.

22. The engineered NK cell of any one of embodiments 1-21, wherein theexpression of FcRγ chain expressed in the cell is undetectable in animmunoblot assay.

23. The engineered NK cell of any of embodiments 1-22, wherein CD16 isexpressed on the surface of the engineered NK cell.

24. The engineered NK cell of any one of embodiments 1-23, wherein theengineered NK cell expresses CD3-zeta (CD3ζ) chain.

25. The engineered NK cell of any one of embodiments 1-24 that isderived from a primary cell obtained from a subject.

26. The engineered NK cell of embodiment 25, wherein the subject ishuman.

27. The engineered NK cell of any one of embodiments 1-24 that isderived from a clonal cell line.

28. The engineered NK cell of embodiment 27, wherein the clonal cellline is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1.

29. The engineered NK cell of any of embodiments 1-28, wherein theengineered NK cell comprises a recombinant or heterologous CD16 geneand/or a recombinant or heterologous CD3-zeta (CD3ζ) chain.

30. The engineered NK cell of embodiment 29, wherein the CD16 comprisesa CD16-activating mutation, a mutation that results in higher affinityto IgG1, a 158V mutation and/or a 158F mutation.

31. The engineered cell of any of embodiments 1-30, wherein theengineered NK cell exhibits increased activity when stimulated throughCD16 compared to the NK cell without the modification.

32. The engineered NK cell of any one of embodiments 1-31, wherein theengineered NK cell has reduced surface expression of NKp46, NKp30,and/or NKp44 compared to the NK cell without the modification.

33. A method of producing an engineered NK cell, comprising geneticallyengineering an NK cell to reduce FcRγ chain expression, activity, and/orsignaling in the cell.

34. The method of embodiment 33, wherein reducing expression comprises:

disrupting or repressing a gene encoding FcRγ chain and/or disrupting orrepressing a gene that results in reduced expression of FcRγ chain inthe NK cell;

disrupting or repressing a gene encoding a protein regulating expressionor activity of FcRγ chain and/or a disrupting or repressing a gene thatresults in reduced expression of a protein regulating expression oractivity of FcRγ chain in the NK cell; and/or

disrupting or repressing a gene encoding a protein involved in FcRγchain-dependent signaling and/or a disrupting or repressing a gene thatresults in reduced expression of a protein involved in FcRγchain-dependent signaling in the NK cell.

35. The method of embodiment 33 or embodiment 34, comprising introducinga deletion, mutation, or insertion into the gene.

36. The method of any one of embodiments 33-35, wherein the gene encodesFcRγ chain.

37. The method of any one of embodiments 33-35 wherein the gene encodesa protein regulating expression or activity of FcRγ chain that is atranscription factor.

38. The method of embodiment 37, wherein the transcription factor isPLZF or HELIOS.

39. The method of any one of embodiments 33-35, wherein the gene encodesa protein involved in FcRγ chain-dependent signaling that is adownstream signaling molecule.

40. The method of embodiment 38, wherein the downstream signalingmolecule is SYK, DAB2 or EAT2.

41. The method of any of embodiments 33-40, wherein disruption orrepression of the gene is effected by introducing an endonuclease thattarget to the gene in the NK cell under conditions that allow disruptionor repression of the gene.

42. The method of embodiment 41, wherein the endonuclease is selectedfrom the group consisting of TAL nucleases, meganucleases, zinc-fingernucleases, Cas9, and Argonaute.

43. The method of any of embodiments 33-40, wherein disruption orrepression is effected by introducing an inhibitory nucleic acid thattargets the gene into the NK cell under conditions that results inrepression of the gene.

44. The method of embodiment 43, wherein the inhibitory nucleic acidmolecule comprises a sequence complementary to the gene encoding FcRγchain.

45. The method of embodiment 43 or embodiment 44, wherein the inhibitorynucleic acid comprises an RNA interfering agent.

46. The method of any one of embodiments 43-45, wherein the nucleic acidis siRNA, shRNA, or miRNA.

47. The method of any one of embodiments 33-46, wherein the reducedexpression is permanent, transient, or inducible.

48. The method of any one of embodiments 33-47, wherein the expression,activity and/or signaling of FcRγ chain is reduced by greater than orgreater than about 50%, 60%, 70%, 80%, 90%, or 95% as compared to theexpression in the NK cell in that is not genetically engineered.

49. The method of any one of embodiments 33-48, wherein the FcRγ chainexpression level is not detectable by an immunoblot assay.

50. The method of any one of embodiments 33-49, comprising, prior to thegenetically engineering, isolating the NK cell from a human subject.

51. The method of embodiment 50, comprising isolating the NK cell fromperipheral blood mononuclear cells (PBMC).

52. The method of embodiment 50 or 51, wherein isolating the NK cellcomprises selecting NK cells from PBMC using a NK cell marker.

53. The method of embodiment 52, wherein the NK cell marker is CD56,Cd161, KIR, NKG2A, NKG2D, NKp30, NKp44, and/or NKp46.

54. The method of any one of embodiments 50-53, further comprisingselecting NK cells that express CD16 and/or CD3ζ.

55. The method of any one of embodiments 50-54, further comprisingselecting NK cells that do not express surface CD3.

56. The method of any one of embodiments 33-49, wherein the NK cell isan NK cell line.

57. The method of embodiment 56, wherein the cell line is NK-92, NK-YS,KHYG-1, NKL, NKG, SNK-6, or IMC-1.

58. The method of embodiment 56 or embodiment 57, further comprisingengineering the NK cell line to express a recombinant or heterologousCD16 and/or CD3ζ.

59. The method of embodiment 58, comprising introducing nucleic acidencoding the CD16 and/or CD3ζ into the NK cell.

60. The method of embodiment 58 or embodiment 59, wherein the CD16 genecontains an activating mutation and/or a mutation that increasesaffinity of CD16 for IgG.

61. The method of any of embodiments 58-60, wherein the CD16 comprises amutation that is 158V.

62. The method of any of embodiments 58-60, wherein the CD16 comprises amutation that is 158F.

63. The method of any of embodiments 58-62, comprising virallytransducing the NK cell with nucleic acid encoding the CD16 and/or CD3ζ.

64. The method of any of embodiments 58-62, comprising transfecting theNK cell with nucleic acid encoding the CD16 and/or CD3ζ.

65. The method of any one of embodiments 58-64, comprising transiently,inducibly, or permanently expressing CD16 or CD3ζ in the NK cell.

66. The method of any one of embodiments 33-65, further comprisingculturing or expanding the engineered NK cells.

67. The method of embodiment 66, comprising culturing the engineered NKcells with feeder cells.

68. The method of embodiment 66 or embodiment 67 comprising culturingthe engineered NK cells with cytokines.

69. An engineered NK cell produced by the methods of any one ofembodiments 33-68.

70. A composition comprising an effective amount of the engineered NKcell of any one of embodiments 1-32, or 69.

71. The composition of embodiment 70, further comprising apharmaceutically acceptable carrier.

72. The composition of embodiment 71, wherein the carrier is a salinesolution, a dextrose solution, or 5% human serum albumin.

73. The composition of any one of embodiments 70-72, wherein thecomposition comprises between 1×10⁵ and 1×10⁸ cells/mL.

74. The composition of any of embodiments 70-73 comprising acryoprotectant.

75. A kit comprising the engineered cell of any one of embodiments 1-32,or 69 or a composition of any one of embodiments 70-74 and an additionalagent for treatment of a disease.

76. The kit of embodiment 75, wherein the additional agent is anantibody or an Fc-fusion protein.

77. The kit of embodiment 76, wherein the antibody recognizes orspecifically binds a tumor associated antigen.

78. The kit of embodiment 77, wherein the tumor associated antigen isCD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74,CD140, EpCAM, CEA, gpA33, mesothelin, α-fetoprotein, Mucin, PDGFR-alpha,TAG-72, CAIX, PSMA, folate-binding protein, scatter factor receptorkinase, a ganglioside, cytokerain, frizzled receptor, VEGF, VEGFR,Integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3,fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-alpha,phosphatidylserine, Syndecan 1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL,FAP, vimentin or tenascin.

79. The kit of any one of embodiments 76-78, wherein the antibody is afull length antibody and/or comprises an Fc domain.

80. A method of treating a condition comprising administering engineeredNK cells of any one of embodiments 1-32, or 69 or a composition of anyone of embodiments 70-74 to an individual in need thereof.

81. The method of embodiment 80, wherein prior to administering theengineered NK cells producing the engineered NK cells by the method ofany of embodiments 33-68.

82. The method of embodiment 80 or embodiment 81, comprisingadministering from or from about 1×10⁸ to 1×10¹⁰ cells/m² to theindividual or administering from or from about 1×10⁶ to 1×10¹⁰ NKcells/kg.

83. The method of any one of embodiments 80-82 further comprisingadministering an additional agent.

84. The method of embodiment 83, wherein the additional agent is anantibody or an Fc-fusion protein.

85. The method of embodiment 84 wherein the antibody recognizes a tumorassociated antigen.

86. The method of embodiment 84 or embodiment 85, wherein the tumorassociated antigen is CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40,CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin,α-fetoprotein, Mucin, PDGFR-alpha, TAG-72, CAIX, PSMA, folate-bindingprotein, scatter factor receptor kinase, a ganglioside, cytokerain,frizzled receptor, VEGF, VEGFR, Integrin αVβ3, integrin α5β1, EGFR,EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R,IGLF2, EPHA3, FR-alpha, phosphatidylserine, Syndecan 1, SLAMF7 (CD319),TRAILR1, TRAILR2, RANKL, FAP, vimentin or tenascin.

87. The method of any one of embodiments 84-86, wherein the antibodycomprises an Fc domain and/or is a full-length antibody.

88. The method of any one of embodiments 83-87, wherein the additionalagent and the engineered NK cells are administered sequentially.

89. The method of embodiment 88, wherein the additional agent isadministered prior to administration of the engineered NK cells.

90. The method of any one of embodiments 83-87, where the additionalagent and the engineered NK cells are administered simultaneously

91. The method of any one of embodiments 80-90, wherein the condition isselected from the group consisting of an inflammatory condition, aninfection, and cancer.

92. The method of embodiment 91, wherein the infection is a viralinfection or a bacterial infection.

93. The method of embodiment 92, wherein the cancer is leukemia orlymphoma.

94. The method of embodiment 92, wherein the cancer comprises a solidtumor.

95. The method of any one of embodiments 80-94, wherein the individualis a human.

96. The method of any one of embodiments 80-95, wherein the engineeredNK cell is allogenic to the individual.

97. The method of any one of embodiments 80-95, wherein the engineeredNK cell is autologous to the subject.

98. The engineered NK cell of any one of embodiments 1-32, wherein thecell is genetically engineered to reduce expression of a NK inhibitoryreceptor.

99. The engineered NK cell of embodiment 98, wherein:

the cell comprises a genetic disruption of a gene encoding a NKinhibitory receptor and/or a genetic disruption resulting in reducedexpression of an NK inhibitory receptor; or

the cell comprises an inhibitory nucleic acid that targets a geneencoding a NK inhibitory receptor and/or that reduces expression of anNK inhibitory receptor.

100. The engineered NK cell of embodiment 98 or embodiment 99, whereinthe inhibitory receptor is NKG2A or KIR2DL1.

101. The method of any one of embodiments 32-68, further comprisinggenetically engineering the NK cell to reduce expression or activity ofan inhibitory receptor.

102. The method of embodiment 101, wherein reducing expression oractivity comprises disrupting or repressing expression of a geneencoding the NK inhibitory receptor or a gene that results in reducedexpression of the NK inhibitory receptor.

103. The method of embodiment 102, wherein the inhibitory receptor isNKG2A or KIR2DL1.

VII. Examples

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Assessment of Cell Killing of Expanded G-NK Cells andConventional NK Cells

25×10⁶ peripheral blood mononuclear cells (PBMCs) from a healthy humandonor were obtained, and fluorescence-activated cell sorting was used toenrich ˜2×10⁵ FcRγ-deficient (g⁻ NK) cells based on certain cell surfacephenotypes. G-NK cells were expanded 250-fold in 14 days by mixing 0.5mL (1×10⁶) of feeder cells (irradiated autologous PBMC stimulated with10 ng/mL OKT3) with 0.5 mL (2×10⁵) of sorted g-NK cells. The expansionwas spiked with 10 ng/mL IL-2 every 2 days and on day 7, expanding g-NKcells were re-fed the cells with irradiated feeders (pre-activated with10 ng/mL OKT3) at a 5:1 feeder: g-NK cell ratio. Conventional NK cellsalso were obtained and expanded.

To assess antibody-directed activity of expanded g-NK cells, serialdilutions of the enriched g-NK cells (E/T ratio ranging from 50:1 to1:1) were incubated in a 96-well plate with ⁵¹Cr-labeled Raji lymphomacells in the presence or absence of anti-CD20 antibody rituximab (5μg/mL). After incubation for 4 hours, antibody dependent cellcytotoxicity (ADCC) was assessed by determining ⁵¹Cr activity per well.As shown in FIG. 1, the results showed that g-NK cells exhibited greaterability to mediate ADCC when co-cultured with rituximab and Rajilymphoma cells than conventional NK cells.

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe engineered cells, compositions and methods described will becomeapparent from the description and teachings herein. Such variations maybe practiced without departing from the true scope and spirit of thedisclosure and are intended to fall within the scope of the presentdisclosure.

What is claimed is:
 1. An engineered natural killer (NK) cell, whereinthe NK cell is genetically engineered to reduce FcRγ chain expression,activity and/or signaling in the cell.
 2. The engineered NK cell ofclaim 1, wherein the engineered NK cell comprises: a genetic disruptionof a gene encoding FcRγ chain and/or a genetic disruption resulting inreduced expression of FcRγ chain in the engineered NK cell; a geneticdisruption of a gene encoding a protein regulating expression oractivity of FcRγ chain and/or a genetic disruption resulting in reducedexpression of a protein regulating expression or activity of FcRγ chain;and/or a genetic disruption of a gene encoding a protein involved inFcRγ chain-dependent signaling and/or a genetic disruption resulting inreduced expression of a protein involved in FcRγ chain-dependentsignaling.
 3. The engineered NK cell of claim 2, wherein the geneticdisruption comprises a deletion, mutation and/or insertion resulting ina premature stop codon in the gene or a frameshift of the open readingframe of the gene.
 4. The engineered NK cell of claim 2 or claim 3,wherein both alleles of the gene encoding FcRγ chain, the gene encodinga protein regulating expression or activity of FcRγ chain and/or thegene encoding a protein involved in FcRγ chain-dependent signaling aredisrupted in the engineered NK cells.
 5. The engineered NK cell of claim1, wherein the engineered NK cell comprises an inhibitory nucleic acidmolecule targeting a gene in the NK cell resulting in reduced expressionof FcRγ chain, reduced expression of a protein regulating expression oractivity of FcRγ chain and/or reduced expression of a protein involvedin FcRγ chain-dependent signaling.
 6. The engineered NK cell of any ofclaims 2-5, wherein the expression of FcRγ chain, a protein regulatingexpression or activity of FcRγ chain and/or a protein involved in FcRγchain-dependent signaling is reduced by greater than or greater thanabout 50%, 60%, 70%, 80%, 90%, or 95% as compared to the expression ofthe protein in the NK cell that is not genetically engineered.
 7. Theengineered NK cell of any of claims 2-6, wherein expression of a proteinregulating expression or activity of FcRγ chain is reduced in theengineered NK cell and the protein is a transcription factor.
 8. Theengineered NK cell of claim 7, wherein the transcription factor is PLZF(ZBTB16) or HELIOS (IKZF2).
 9. The engineered NK cell of any of claims2-6, wherein the expression of a protein involved in FcRγchain-dependent signaling is reduced in the engineered NK cell and theprotein is a downstream signaling molecule.
 10. The engineered NK cellsof claim 9, wherein the downstream signaling molecule is SYK, DAB2 orEAT-2.
 11. The engineered NK cell of any of claims 2-6, whereinexpression of FcRγ chain is reduced in the engineered cell.
 12. Anengineered NK cell comprising a genetic disruption in a gene encodingFcRγ chain, wherein expression of FcRγ is reduced in the cell.
 13. Theengineered NK cell of claim 12, wherein the genetic disruption comprisesa deletion, mutation and/or insertion resulting in a premature stopcodon in the gene or a frameshift of the open reading frame of the gene.14. The engineered NK cell of claim 12 or claim 13, wherein both allelesof the gene encoding FcRγ chain are disrupted in the genome of theengineered NK cell.
 15. The engineered NK cell of any of claims 1, 5 or6, wherein the engineered NK cell comprises an inhibitory nucleic acidmolecule that targets a gene encoding FcRγ chain.
 16. The engineered NKcell of claim 15, wherein the inhibitory nucleic acid molecule comprisesa sequence complementary to the gene encoding FcRγ chain.
 17. Theengineered NK cell of any of claims 11-16, wherein the expression ofFcRγ chain is reduced by greater than or greater than about 50%, 60%,70%, 80%, 90%, or 95% as compared to the expression in the NK cell thatis not genetically engineered.
 18. The engineered NK cell of any ofclaims 5-6 and 15-17, wherein the inhibitory nucleic acid comprises anRNA interfering agent.
 19. The engineered NK cell of any of claims 5-6and 15-18, wherein the inhibitory nucleic acid comprises siRNA, shRNA,or miRNA.
 20. The engineered NK cell of any of claims 1-19, wherein thereduced expression, activity and/or signaling of FcRγ is permanent,transient or inducible.
 21. The engineered NK cell of any of claims1-20, wherein the expression, activity and/or signaling of FcRγ chain isreduced by greater than or greater than about 50%, 60%, 70%, 80%, 90%,or 95% as compared to the expression, activity and/or signaling in theNK cell that is not genetically engineered.
 22. The engineered NK cellof any one of claims 1-21, wherein the expression of FcRγ chainexpressed in the cell is undetectable in an immunoblot assay.
 23. Theengineered NK cell of any of claims 1-22, wherein CD16 is expressed onthe surface of the engineered NK cell.
 24. The engineered NK cell of anyone of claims 1-23, wherein the engineered NK cell expresses CD3-zeta(CD3ζ) chain.
 25. The engineered NK cell of any one of claims 1-24 thatis derived from a primary cell obtained from a subject.
 26. Theengineered NK cell of claim 25, wherein the subject is human.
 27. Theengineered NK cell of any one of claims 1-24 that is derived from aclonal cell line.
 28. The engineered NK cell of claim 27, wherein theclonal cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, or IMC-1. 29.The engineered NK cell of any of claims 1-28, wherein the engineered NKcell comprises a recombinant or heterologous CD16 gene and/or arecombinant or heterologous CD3-zeta (CD3ζ) chain.
 30. The engineered NKcell of claim 29, wherein the CD16 comprises a CD16-activating mutation,a mutation that results in higher affinity to IgG1, a 158V mutationand/or a 158F mutation.
 31. The engineered cell of any of claims 1-30,wherein the engineered NK cell exhibits increased activity whenstimulated through CD16 compared to the NK cell without themodification.
 32. The engineered NK cell of any one of claims 1-31,wherein the engineered NK cell has reduced surface expression of NKp46,NKp30, and/or NKp44 compared to the NK cell without the modification.33. A method of producing an engineered NK cell, comprising geneticallyengineering an NK cell to reduce FcRγ chain expression, activity, and/orsignaling in the cell.
 34. The method of claim 33, wherein reducingexpression comprises: disrupting or repressing a gene encoding FcRγchain and/or disrupting or repressing a gene that results in reducedexpression of FcRγ chain in the NK cell; disrupting or repressing a geneencoding a protein regulating expression or activity of FcRγ chainand/or a disrupting or repressing a gene that results in reducedexpression of a protein regulating expression or activity of FcRγ chainin the NK cell; and/or disrupting or repressing a gene encoding aprotein involved in FcRγ chain-dependent signaling and/or a disruptingor repressing a gene that results in reduced expression of a proteininvolved in FcRγ chain-dependent signaling in the NK cell.
 35. Themethod of claim 33 or claim 34, comprising introducing a deletion,mutation, or insertion into the gene.
 36. The method of any one ofclaims 33-35, wherein the gene encodes FcRγ chain.
 37. The method of anyone of claims 33-35 wherein the gene encodes a protein regulatingexpression or activity of FcRγ chain that is a transcription factor. 38.The method of claim 37, wherein the transcription factor is PLZF orHELIOS.
 39. The method of any one of claims 33-35, wherein the geneencodes a protein involved in FcRγ chain-dependent signaling that is adownstream signaling molecule.
 40. The method of claim 38, wherein thedownstream signaling molecule is SYK, DAB2 or EAT2.
 41. The method ofany of claims 33-40, wherein disruption or repression of the gene iseffected by introducing an endonuclease that target to the gene in theNK cell under conditions that allow disruption or repression of thegene.
 42. The method of claim 41, wherein the endonuclease is selectedfrom the group consisting of TAL nucleases, meganucleases, zinc-fingernucleases, Cas9, and Argonaute.
 43. The method of any of claims 33-40,wherein disruption or repression is effected by introducing aninhibitory nucleic acid that targets the gene into the NK cell underconditions that results in repression of the gene.
 44. The method ofclaim 43, wherein the inhibitory nucleic acid molecule comprises asequence complementary to the gene encoding FcRγ chain.
 45. The methodof claim 43 or claim 44, wherein the inhibitory nucleic acid comprisesan RNA interfering agent.
 46. The method of any one of claims 43-45,wherein the nucleic acid is siRNA, shRNA, or miRNA.
 47. The method ofany one of claims 33-46, wherein the reduced expression is permanent,transient, or inducible.
 48. The method of any one of claims 33-47,wherein the expression, activity and/or signaling of FcRγ chain isreduced by greater than or greater than about 50%, 60%, 70%, 80%, 90%,or 95% as compared to the expression in the NK cell in that is notgenetically engineered.
 49. The method of any one of claims 33-48,wherein the FcRγ chain expression level is not detectable by animmunoblot assay.
 50. The method of any one of claims 33-49, comprising,prior to the genetically engineering, isolating the NK cell from a humansubject.
 51. The method of claim 50, comprising isolating the NK cellfrom peripheral blood mononuclear cells (PBMC).
 52. The method of claim50 or 51, wherein isolating the NK cell comprises selecting NK cellsfrom PBMC using a NK cell marker.
 53. The method of claim 52, whereinthe NK cell marker is CD56, Cd161, KIR, NKG2A, NKG2D, NKp30, NKp44,and/or NKp46.
 54. The method of any one of claims 50-53, furthercomprising selecting NK cells that express CD16 and/or CD3.
 55. Themethod of any one of claims 50-54, further comprising selecting NK cellsthat do not express surface CD3.
 56. The method of any one of claims33-49, wherein the NK cell is an NK cell line.
 57. The method of claim56, wherein the cell line is NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, orIMC-1.
 58. The method of claim 56 or claim 57, further comprisingengineering the NK cell line to express a recombinant or heterologousCD16 and/or CD3ζ.
 59. The method of claim 58, comprising introducingnucleic acid encoding the CD16 and/or CD3ζ into the NK cell.
 60. Themethod of claim 58 or claim 59, wherein the CD16 gene contains anactivating mutation and/or a mutation that increases affinity of CD16for IgG.
 61. The method of any of claims 58-60, wherein the CD16comprises a mutation that is 158V.
 62. The method of any of claims58-60, wherein CD16 comprises a mutation that is 158F.
 63. The method ofany of claims 58-62, comprising virally transducing the NK cell withnucleic acid encoding the CD16 and/or CD3ζ.
 64. The method of any ofclaims 58-62, comprising transfecting the NK cell with nucleic acidencoding the CD16 and/or CD3ζ.
 65. The method of any one of claims58-64, comprising transiently, inducibly, or permanently expressing CD16or CD3ζ in the NK cell.
 66. The method of any one of claims 33-65,further comprising culturing or expanding the engineered NK cells. 67.The method of claim 66, comprising culturing the engineered NK cellswith feeder cells.
 68. The method of claim 66 or claim 67 comprisingculturing the engineered NK cells with cytokines.
 69. An engineered NKcell produced by the methods of any one of claims 33-68.
 70. Acomposition comprising an effective amount of the engineered NK cell ofany one of claims 1-32, or
 69. 71. The composition of claim 70, furthercomprising a pharmaceutically acceptable carrier.
 72. The composition ofclaim 71, wherein the carrier is a saline solution, a dextrose solution,or 5% human serum albumin.
 73. The composition of any one of claims70-72, wherein the composition comprises between 1×10⁵ and 1×10⁸cells/mL.
 74. The composition of any of claims 70-73 comprising acryoprotectant.
 75. A kit comprising the engineered cell of any one ofclaims 1-32, or 69 or a composition of any one of claims 70-74 and anadditional agent for treatment of a disease.
 76. The kit of claim 75,wherein the additional agent is an antibody or an Fc-fusion protein. 77.The kit of claim 76, wherein the antibody recognizes or specificallybinds a tumor associated antigen.
 78. The kit of claim 77, wherein thetumor associated antigen is CD19, CD20, CD22, CD30, CD33, CD37, CD38,CD40, CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin,α-fetoprotein, Mucin, PDGFR-alpha, TAG-72, CAIX, PSMA, folate-bindingprotein, scatter factor receptor kinase, a ganglioside, cytokerain,frizzled receptor, VEGF, VEGFR, Integrin αVβ3, integrin α5β1, EGFR,EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R,IGLF2, EPHA3, FR-alpha, phosphatidylserine, Syndecan 1, SLAMF7 (CD319),TRAILR1, TRAILR2, RANKL, FAP, vimentin or tenascin.
 79. The kit of anyone of claims 76-78, wherein the antibody is a full length antibodyand/or comprises an Fc domain.
 80. A method of treating a conditioncomprising administering engineered NK cells of any one of claims 1-32,or 69 or a composition of any one of claims 70-74 to an individual inneed thereof.
 81. The method of claim 80, wherein prior to administeringthe engineered NK cells producing the engineered NK cells by the methodof any of claims 33-68.
 82. The method of claim 80 or claim 81,comprising administering from or from about 1×10⁸ to 1×10¹⁰ cells/m² tothe individual or administering from or from about 1×10⁶ to 1×10¹⁰ NKcells/kg.
 83. The method of any one of claims 80-82 further comprisingadministering an additional agent.
 84. The method of claim 83, whereinthe additional agent is an antibody or an Fc-fusion protein.
 85. Themethod of claim 84 wherein the antibody recognizes a tumor associatedantigen.
 86. The method of claim 84 or claim 85, wherein the tumorassociated antigen is CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40,CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin,α-fetoprotein, Mucin, PDGFR-alpha, TAG-72, CAIX, PSMA, folate-bindingprotein, scatter factor receptor kinase, a ganglioside, cytokerain,frizzled receptor, VEGF, VEGFR, Integrin αVβ3, integrin α5β1, EGFR,EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R,IGLF2, EPHA3, FR-alpha, phosphatidylserine, Syndecan 1, SLAMF7 (CD319),TRAILR1, TRAILR2, RANKL, FAP, vimentin or tenascin.
 87. The method ofany one of claims 84-86, wherein the antibody comprises an Fc domainand/or is a full-length antibody.
 88. The method of any one of claims83-87, wherein the additional agent and the engineered NK cells areadministered sequentially.
 89. The method of claim 88, wherein theadditional agent is administered prior to administration of theengineered NK cells.
 90. The method of any one of claims 83-87, wherethe additional agent and the engineered NK cells are administeredsimultaneously
 91. The method of any one of claims 80-90, wherein thecondition is selected from the group consisting of an inflammatorycondition, an infection, and cancer.
 92. The method of claim 91, whereinthe infection is a viral infection or a bacterial infection.
 93. Themethod of claim 92, wherein the cancer is leukemia or lymphoma.
 94. Themethod of claim 92, wherein the cancer comprises a solid tumor.
 95. Themethod of any one of claims 80-94, wherein the individual is a human.96. The method of any one of claims 80-95, wherein the engineered NKcell is allogenic to the individual.
 97. The method of any one of claims80-95, wherein the engineered NK cell is autologous to the subject. 98.The engineered NK cell of any one of claims 1-32, wherein the cell isgenetically engineered to reduce expression of a NK inhibitory receptor.99. The engineered NK cell of claim 98, wherein: the cell comprises agenetic disruption of a gene encoding a NK inhibitory receptor and/or agenetic disruption resulting in reduced expression of an NK inhibitoryreceptor; or the cell comprises an inhibitory nucleic acid that targetsa gene encoding a NK inhibitory receptor and/or that reduces expressionof an NK inhibitory receptor.
 100. The engineered NK cell of claim 98 orclaim 99, wherein the inhibitory receptor is NKG2A or KIR2DL1.
 101. Themethod of any one of claims 32-68, further comprising geneticallyengineering the NK cell to reduce expression or activity of aninhibitory receptor.
 102. The method of claim 101, wherein reducingexpression or activity comprises disrupting or repressing expression ofa gene encoding the NK inhibitory receptor or a gene that results inreduced expression of the NK inhibitory receptor.
 103. The method ofclaim 102, wherein the inhibitory receptor is NKG2A or KIR2DL1.